Nucleic acid promoter fragment isolated from a plant tryptophan synthase alpha subunit (trpA) gene

ABSTRACT

DNA sequences optimized for expression in plants are disclosed. The DNA sequences preferably encode for an insecticidal polypeptides, particularly insecticidal proteins from Bacillus thuringiensis. Plant promoters, particular tissue-specific and tissue-preferred promoters are also provided. Additionally disclosed are transformation vectors comprising said DNA sequences. The transformation vectors demonstrate high levels of insecticidal activity when transformed into maize.

This is a divisional application of Ser. No. 07/951,715, filed Sep. 25,1992 now U.S. Pat. No. 5,625,136, which is a continuation-in-part ofSer. No. 07/772,027, filed Oct. 4, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates to DNA sequences encoding insecticidalproteins, and expression of these sequences in plants.

BACKGROUND OF THE INVENTION

Expression of the insecticidal protein (IP) genes derived from Bacillusthuringiensis (Bt) in plants has proven extremely difficult. Attemptshave been made to express chimeric promoter/Bt IP gene combinations inplants. Typically, only low levels of protein have been obtained intransgenic plants. See, for example, Vaeck et al., Nature 328:33-37,1987; Barton et al., Plant Physiol. 85:1103-1109, 1987; Fischoff et al.,Bio/Technology 5:807-813, 1987.

One postulated explanation for the cause of low expression is thatfortuitious transcription processing sites produce aberrant forms of BtIP mRNA transcript. These aberrantly processed transcripts arenon-functional in a plant, in terms of producing an insecticidalprotein. Possible processing sites include polyadenylation sites, intronsplicing sites, transcriptional termination signals and transportsignals. Most genes do not contain sites that will deleteriously affectgene expression in that gene's normal host organism. However, thefortuitous occurrence of such processing sites in a coding region mightcomplicate the expression of that gene in transgenic hosts. For example,the coding region for the Bt insecticidal crystal protein gene derivedfrom Bacillus thuringiensis strain kurstaki (GENBANK BTHKURHD, accessionM15271, B. thuringiensis var. kurstaki, HD-1; Geiser et al. Gene48:109-118 (1986)) as derived directly from Bacillus thuringiensis,might contain sites which prevent this gene from being properlyprocessed in plants.

Further difficulties exist when attempting to express Bacillusthuringiensis protein in an organism such as a plant. It has beendiscovered that the codon usage of a native Bt IP gene is significantlydifferent from that which is typical of a plant gene. In particular, thecodon usage of a native Bt IP gene is very different from that of amaize gene. As a result, the mRNA from this gene may not be efficientlyutilized. Codon usage might influence the expression of genes at thelevel of translation or transcription or mRNA processing. To optimize aninsecticidal gene for expression in plants, attempts have been made toalter the gene to resemble, as much as possible, genes naturallycontained within the host plant to be transformed.

Adang et al., EP 0359472 (1990), relates to a synthetic Bacillusthuringiensis tenebrionis (Btt) gene which is 85% homologous to thenative Btt gene and which is designed to have an A+T contentapproximating that found in plants in general. Table 1 of Adang et al.show the codon sequence of a synthetic Btt gene which was made toresemble more closely the normal codon distribution of dicot genes.Adang et al. state that a synthetic gene coding for IP can be optimizedfor enhanced expression in monocot plants through similar methods,presenting the frequency of codon usage of highly expressed monocotproteins in Table 1. At page 9, Adang et al. state that the syntheticBtt gene is designed to have an A+T content of 55% (and, by implication,a G+C content of 45%). At page 20, Adang et al. disclose that thesynthetic gene is designed by altering individual amino acid codons inthe native Bt gene to reflect the overall distribution of codonspreferred by dicot genes for each amino acid within the coding region ofthe gene. Adang et al. further state that only some of the native Bttgene codons will be replaced by the most preferred plant codon for eachamino acid, such that the overall distribution of codons used in dicotproteins is preserved.

Fischhoff et al., EP 0 385 962 (1990), relates to plant genes encodingthe crystal protein toxin of Bacillus thuringiensis. At table V,Fischhoff et al. disclose percent usages for codons for each amino acid.At page 8, Fischoff et al. suggest modifying a native Bt gene by removalof putative polyadenylation signals and ATTTA sequences. Fischoff et al.further suggest scanning the native Bt gene sequence for regions withgreater than four consecutive adenine or thymine nucleotides to identifyputative plant polyadenylation signals. Fischoff et al. state that thenucleotide sequence should be altered if more than one putativepolyadenylation signal is identified within ten nucleotides of eachother. At page 9, Fischoff et al. state that efforts should be made toselect codons to preferably adjust the G+C content to about 50%.

Perlak et al., PNAS USA, 88:3324-3328 (1991), relates to modified codingsequences of the Bacillus thuringiensis cryIA(b) gene, similar to thoseshown in Fischoff et al. As shown in table 1 at page 3325, the partiallymodified cryIA(b) gene of Perlak et al. is approximately 96% homologousto the native cryIA(b) gene (1681 of 1743 nucleotides), with a G+Ccontent of 41%, number of plant polyadenylation signal sequences (PPSS)reduced from 18 to 7 and number of ATTTA sequences reduced from 13 to 7.The fully modified cryIA(b) gene of Perlak et al. is disclosed to befully synthetic (page 3325, column 1). This gene is approximately 79%homologous to the native cryIA(b) gene (1455 of 1845 nucleotides), witha G+C content of 49%, number of plant polyadenylation signal sequences(PPSS) reduced to 1 and all ATTTA sequences removed.

Barton et al., EP 0431 829 (1991), relates to the expression ofinsecticidal toxins in plants. At column 10, Barton et al. describe theconstruction of a synthetic AaIT insect toxin gene encoding a scorpiontoxin using the most preferred codon for each amino acid according tothe chart shown in FIG. 1 of the document.

SUMMARY OF THE INVENTION

The present invention is drawn to methods for enhancing expression ofheterologous genes in plant cells. Generally, a gene or coding region ofinterest is constructed to provide a plant specific preferred codonsequence. In this manner, codon usage for a particular protein isaltered to increase expression in a particular plant. Such plantoptimized coding sequences can be operably linked to promoters capableof directing expression of the coding sequence in a plant cell.

Specifically, it is one of the objects of the present invention toprovide synthetic insecticidal protein genes which have been optimizedfor expression in plants.

It is another object of the present invention to provide synthetic Btinsecticidal protein genes to maximize the expression of Bt proteins ina plant, preferably in a maize plant. It is one feature of the presentinvention that a synthetic Bt IP gene is constructed using the mostpreferred maize codons, except for alterations necessary to provideligation sites for construction of the full synthetic gene.

According to the above objects, we have synthesized Bt insecticidalcrystal protein genes in which the codon usage has been altered in orderto increase expression in plants, particularly maize. However, ratherthan alter the codon usage to resemble a maize gene in terms of overallcodon distribution, we have optimized the codon usage by using thecodons which are most preferred in maize (maize preferred codons) in thesynthesis of the synthetic gene. The optimized maize preferred codonusage is effective for expression of high levels of the Bt insecticidalprotein. This might be the result of maximizing the amount of Btinsecticidal protein translated from a given population of messengerRNAs. The synthesis of a Bt IP gene using maize preferred codons alsotends to eliminate fortuitous processing sites that might occur in thenative coding sequence. The expression of this synthetic gene issignificantly higher in maize cells than that of the native IP Bt gene.

Preferred synthetic, maize optimized DNA sequences of the presentinvention derive from the protein encoded by the cryIA(b) gene inBacillus thuringiensis var. kurstaki, HD-1; Geiser et al., Gene,48:109-118 (1986) or the cryIB gene (AKA Crya4 gene) described byBrizzard and Whiteley, Nuc. Acids. Res., 16:2723 (1988). The DNAsequence of the native kurstaki HD-1 cryIA(b) gene is shown as SEQ IDNO: 1. These proteins are active against various lepidopteran insects,including Ostrinia nubilalis, the European Corn Borer.

While the present invention has been exemplified by the synthesis ofmaize optimized Bt protein genes, it is recognized that the method canbe utilized to optimize expression of any protein in plants.

The instant optimized genes can be fused with a variety of promoters,including constitutive, inducible, temporally regulated, developmentallyregulated, tissue-preferred and tissue-specific promoters to preparerecombinant DNA molecules, i.e., chimeric genes. The maize optimizedgene (coding sequence) provides substantially higher levels ofexpression in a transformed plant, when compared with a non-maizeoptimized gene. Accordingly, plants resistant to Coleopteran orLepidopteran pests, such as European corn borer and sugarcane borer, canbe produced.

It is another object of the present invention to providetissue-preferred and tissue-specific promoters which drive theexpression of an operatively associated structural gene of interest in aspecific part or parts of a plant to the substantial exclusion of otherparts.

It is another object of the present invention to provide pith-preferredpromoters. By "pith-preferred," it is intended that the promoter iscapable of directing the expression of an operatively associatedstructural gene in greater abundance in the pith of a plant than in theroots, outer sheath, and brace roots, and with substantially noexpression in seed.

It is yet another object of this invention to provide pollen-specificpromoters. By "pollen-specific," it is intended that the promoter iscapable of directing the expression of an operatively associatedstructural gene of interest substantially exclusively in the pollen of aplant, with negligible expression in any other plant part. By"negligible," it is meant functionally insignificant.

It is yet another object of the present invention to provide recombinantDNA molecules comprising a tissue-preferred promoter or tissue-specificpromoter operably associated or linked to a structural gene of interest,particularly a structural gene encoding an insecticidal protein, andexpression of the recombinant molecule in a plant.

It is a further object of the present invention to provide transgenicplants which express at least one structural gene of interestoperatively in a tissue-preferred or tissue-specific expression pattern.

In one specific embodiment of the invention disclosed and claimedherein, the tissue-preferred or tissue-specific promoter is operablylinked to a structural gene encoding an insecticidal protein, and aplant is stably transformed with at least one such recombinant molecule.The resultant plant will be resistant to particular insects which feedon those parts of the plant in which the gene(s) is(are) expressed.Preferred structural genes encode B.t. insecticidal proteins. Morepreferred are maize optimized B.t. IP genes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a comparison of the full-length native Bt cryIA(b) gene(BTHKURHD; SEQ ID NO: 1), a full-length synthetic maize optimized BtcryIA(b) gene (flsynbt.fin; SEQ ID NO: 4) and a truncated syntheticmaize optimized Bt cryIA(b) gene (bssyn; SEQ ID NO: 3). This figureshows that the full-length synthetic maize optimized cryIA(b) genesequence matches that of the native cryIA(b) gene at about 2354 out of3468 nucleotides (approximately 68% homology).

FIG. 2 is a comparison of the truncated native Bt cryIA(b) gene(nucleotides 1 to 1947 of BTHKURHD; SEQ ID NO: 1) and a truncatedsynthetic maize optimized Bt gene (bssyn; SEQ ID NO: 3). This figureshows that the truncated synthetic maize optimized cryIA(b) genesequence matches that of the native cryIA(b) gene at about 1278 out of1947 nucleotides (approximately 66% homology).

FIG. 3 is a comparison of the pure maize optimized Bt gene sequence(syn1T.mze; SEQ ID NO: 2) with a truncated synthetic maize optimized Btgene (bssyn; SEQ ID NO: 3) and a full-length synthetic maize optimizedBt gene modified to include restriction sites for facilitatingconstruction of the gene (synful.mod; SEQ ID NO: 4). This figure showsthat the truncated synthetic maize optimized cryIA(b) gene sequencematches that of the pure maize optimized cryIA(b) gene at 1913 out of1947 nucleotides (approximately 98% homology).

FIG. 4 is a comparison of a native truncated Bt cryIA(b) gene(nucleotides 1 to 1845 of BTHKURHD SEQ ID NO: 1) with a truncatedsynthetic cryIA(b) gene described in Perlak et al., PNAS USA,88:3324-3328 (1991) (PMONBT SEQ ID NO: 5) and a truncated syntheticmaize optimized Bt gene (bssyn SEQ ID NO: 3). This figure shows that thePMONBT gene sequence matches that of the native cryIA(b) gene at about1453 out of 1845 nucleotides (approximately 79% homology), while thetruncated synthetic maize optimized Bt cryIA(b) gene matches the nativecryIA(b) gene at about 1209 out of 1845 nucleotides (approximately 66%homology).

FIG. 5 is a comparison of a truncated synthetic cryIA(b) gene describedin Perlak et al., PNAS USA, 88:3324-3328 (1991) (PMONBT SEQ ID NO: 5)and a truncated synthetic maize optimized Bt cryIA(b) gene (bssyn SEQ IDNO: 3). This figure shows that the PMONBT gene sequence matches that ofthe truncated synthetic maize optimized Bt cryIA(b) gene at about 1410out of 1845 nucleotides (approximately 77% homology).

FIG. 6 is a full-length, maize optimized CryIB gene (SEQ ID NO: 6)encoding the CryIB protein (SEQ ID NO: 7).

FIG. 7 is a full-length, hybrid, partially maize optimized DNA sequenceof a CryIA(b) gene (SEQ ID NO: 8) which is contained in pCIB4434. Thesynthetic region is from nucleotides 1-1938 (amino acids 1-646 SEQ IDNO: 9), and the native region is from nucleotides 1939-3468 (amino acids647-1155 SEQ ID NO: 9). The fusion point between the synthetic andnative coding sequences is indicated by a slash (/) in the sequence.

FIG. 8 is a map of pCIB4434.

FIG. 9 is a full-length, hybrid, maize optimized DNA sequence (SEQ IDNO: 10) encoding a heat stable CryIA(b) protein (SEQ ID NO: 11),contained in pCIB5511.

FIG. 10 is a map of pCIB5511.

FIG. 11 is a full-length, hybrid, maize optimized DNA sequence (SEQ IDNO: 12) encoding a heat stable CryIA(b) protein (SEQ ID NO: 13),contained in pCIB5512.

FIG. 12 is a map of pCIB5512.

FIG. 13 is a full-length, maize optimized DNA sequence (SEQ ID NO: 14)encoding a heat stable CryIA(b) protein (SEQ ID NO: 15), contained inpCIB5513.

FIG. 14 is a map of pCIB5513.

FIG. 15 is a full-length, maize optimized DNA sequence (SEQ ID NO: 16)encoding a heat-stable CryIA(b) protein (SEQ ID NO: 17), contained inpCIB5514.

FIG. 16 is a map of pCIB5514.

FIG. 17 is a map of pCIB4418.

FIG. 18 is a map of pCIB4420.

FIG. 19 is a map of pCIB4429.

FIG. 20 is a map of pCIB4431.

FIG. 21 is a map of pCIB4428.

FIG. 22 is a map of pCIB4430.

FIG. 23A is a table containing data of cryIA(b) protein levels intransgenic maize.

FIG. 23B is a table which summarizes results of bioassays of Ostriniaand Diatraea on leaf material from maize progeny containing a maizeoptimized CryIA(b) gene.

FIG. 23C is a table containing data of cryIA(b) protein levels intransgenic maize.

FIG. 23D is a table which summarizes the results of bioassays ofOstrinia and Diatraea on leaf material from maize progeny containing asynthetic Bt. maize gene operably linked to a pith promoter.

FIG. 23E is a table containing data on expression of the cryIA(b) genein transgenic maize using the pith-preferred promoter. Leaf samples fromsmall plantlets transformed with pCIB4433 using procedures describedelsewhere were analyzed for the presence of the cryIA(b) protein usingELISA. All plants expressing cryIA(b) were found to be insecticidal inthe standard European corn borer bioassay. Note that the pith-preferredpromoter has a low, but detectable level of expression in leaf tissue ofmaize. Detection of CryIA(b) protein is consistent with this pattern ofexpression.

FIG. 24 is a complete genomic DNA sequence (SEQ ID NO: 18) encoding amaize tryptophan synthase-alpha subunit (TrpA) protein (SEQ ID NO: 19).Introns, exons, transcription and translation starts, start and stop ofcDNA are shown. $=start and end of cDNA; +1=transcription start;73*******=primer extension primer; +1=start of translation; +++=stopcodon; bp 1495-99=CCAAT Box; bp 1593-1598=TATAA Box; bp 3720-3725=poly Aaddition site;

# above underlined sequences are PCR primers.

FIGS. 25A, 25B, 25C and 25D are Northern blot analyses which showdifferential expression of the maize TrpA subunit gene in maize tissueat 2 hour, 4 hour, 18 hour, and 48 hour intervals, respectively, at -80°C. with DuPont Cronex intensifying screens. P=pith; C=cob; BR=braceroots; ES=ear shank; LP=lower pith; MP=middle pith; UP=upper pith;S=seed; L=leaf; R=root; SH=sheath; and P(upper left)=total pith.

FIG. 26 is a Northern blot analysis, the two left lanes of which showthe maize TrpA gene expression in the leaf (L) and pith (P) of Funkinbred lines 211D and 5N984. The five right lanes indicate the absenceof expression in Funk 211D seed total RNA. S(1, 2,3, 4 and 5)=seed at 1,2, 3, 4 and 5 weeks post pollenation. L=leaf; P=pith; S#=seed # weekspost pollenation.

FIG. 27 is a Southern blot analysis of genomic DNA Funk line 211D,probed with maize TrpA cDNA 8-2 (pCIB5600), wherein B denotes BamHI, Edenotes EcoRI, EV denotes EcoRV, H denotes HINDIII, and S denotes SacI.1X, 5X and 10X denote reconstructed gene copy equivalents.

FIG. 28A is a primer extension analysis which shows the transcriptionalstart of the maize TrpA subunit gene and sequencing ladder at a 1 hourexposure against film at -80° C. with Dupont Cronex intensifyingscreens. Lane+1 and +2 are 1X+0.5X samples of primer extension reaction.

FIG. 28B is an analysis of RNase protection from +2 bp to +387 bp atannealing temperatures of 42° C., 48° C. and 54° C., at a 16 hourexposure against film at -80° C. with DuPont Cronex intensifyingscreens.

FIG. 29 is A map of the original Type II pollen-specific cDNA clone. Thesubcloning of the three EcoRI fragments into pBluescript vectors tocreate pCIB3168, pCIB3169 and II-.6 is illustrated.

FIG. 30 shows the DNA sequence of the maize pollen-specific calciumdependent protein kinase gene cDNA (SEQ ID NO: 20), as contained in the1.0 kb and 0.5 kb fragments of the original Type II cDNA clone. TheEcoRI site that divides the 1.0 kb and 0.5 kb fragments is indicated.This cDNA is not full length, as the mRNA start site maps 490 bpupstream of the end of the cDNA clone. The translated protein isdisclosed as SEQ ID NO: 21.

FIG. 31 illustrates the tissue-specific expression of the pollen CDPKmRNA. RNA from the indicated maize 211D tissues was denatured,electrophoresed on an agarose gel, transferred to nitrocellulose, andprobed with the pollen CDPK cDNA 0.5 kb fragment. The mRNA is detectableonly in the pollen, where a strong signal is seen.

FIG. 32 is an amino acid sequence (sequence line 1, amino acids 13 to307 of SEQ ID NO: 22) comparison of the pollen CDPK derived proteinsequence and the rat calmodulin-dependent protein kinase 2 proteinsequence (sequence line 3; SEQ ID NO: 23) disclosed in Tobimatsu et al.,J. Biol. Chem. 263:16082-16086 (1988). The Align program of the DNAstarsoftware package was used to evaluate the sequences. The homology toprotein kinases occurs in the 5' two thirds of the gene, i.e. in the 1.0kb fragment.

FIG. 33 is an amino acid sequence comparison of the pollen CDPK derivedprotein sequence (sequence line 1; amino acids 311 to 450 of SEQ ID NO:22) and the human calmodulin protein sequence (sequence line 3; SEQ IDNO: 24) disclosed in Fischer et al., J. Biol. Chem. 263:17055-17062(1988). The homology to calmodulin occurs in the 3' one third of thegene, i.e. in the 0.5 kb fragment.

FIG. 34 is an amino acid sequence comparison of the pollen CDPK derivedprotein sequence (sequence line 1; SEQ ID NO: 22) and soybean CDPK (SEQID NO: 25). The homology occurs over the entire gene.

FIG. 35 illustrates the sequence of the maize pollen-specific CDPK gene(SEQ ID NO: 26). 1.4 kb of sequence prior to the mRNA start site isshown. The positions of the seven exons and six introns are depictedunder the corresponding DNA sequence. The site of polyadenylation in thecDNA clone is indicated.

FIG. 36 is a map of pCIB4433.

FIG. 37 is a full-length, hybrid, maize-optimized DNA sequence (SEQ IDNO: 27) encoding a heat stable cryIA(b) protein (SEQ ID NO: 28).

FIG. 38 is a map of pCIB5515.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the DNA sequence of a full-length native Bt cryIA(b)gene.

SEQ ID NO: 2 is the DNA sequence of a full-length pure maize optimizedsynthetic Bt cryIA(b) gene.

SEQ ID NO: 3 is the DNA sequence of an approximately 2 Kb truncatedsynthetic maize optimized Bt cryIA(b) gene.

SEQ ID NO: 4 is the DNA sequence of a full-length synthetic maizeoptimized Bt cryIA(b) gene.

SEQ ID NO: 5 is the DNA sequence of an approximately 2 Kb synthetic Btgene according to Perlak et al.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided in order to provide clarity withrespect to the terms as they are used in the specification and claims todescribe the present invention.

Maize preferred codon: Preferred codon refers to the preferenceexhibited by a specific host cell in the usage of nucleotide codons tospecify a given amino acid. The preferred codon for an amino acid for aparticular host is the single codon which most frequently encodes thatamino acid in that host. The maize preferred codon for a particularamino acid may be derived from known gene sequences from maize. Forexample, maize codon usage for 28 genes from maize plants are listed inTable 4 of Murray et al., Nucleic Acids Research, 17:477-498 (1989), thedisclosure of which is incorporated herein by reference. For instance,the maize preferred codon for alanine is GCC, since, according to pooledsequences of 26 maize genes in Murray et al., supra, that codon encodesalanine 36% of the time, compared to GCG (24%), GCA (13%), and GCT(27%).

Pure maize optimized sequence: An optimized gene or DNA sequence refersto a gene in which the nucleotide sequence of a native gene has beenmodified in order to utilize preferred codons for maize. For example, asynthetic maize optimized Bt cryIA(b) gene is one wherein the nucleotidesequence of the native Bt cryIA(b) gene has been modified such that thecodons used are the maize preferred codons, as described above. A puremaize optimized gene is one in which the nucleotide sequence comprises100 percent of the maize preferred codon sequences for a particularpolypeptide. For example, the pure maize optimized Bt cryIA(b) gene isone in which the nucleotide sequence comprises 100 percent maizepreferred codon sequences and encodes a polypeptide with the same aminoacid sequence as that produced by the native Bt cryIA(b) gene. The purenucleotide sequence of the optimized gene may be varied to permitmanipulation of the gene, such as by altering a nucleotide to create oreliminate restriction sites. The pure nucleotide sequence of theoptimized gene may also be varied to eliminate potentially deleteriousprocessing sites, such as potential polyadenylation sites or intronrecognition sites.

It is recognized that "partially maize optimized," sequences may also beutilized. By partially maize optimized, it is meant that the codingregion of the gene is a chimeric (hybrid), being comprised of sequencesderived from a native insecticidal gene and sequences which have beenoptimized for expression in maize. A partially optimized gene expressesthe insecticidal protein at a level sufficient to control insect pests,and such expression is at a higher level than achieved using nativesequences only. Partially maize optimized sequences include those whichcontain at least about 5% optimized sequences.

Full-length Bt Genes: Refers to DNA sequences comprising the fullnucleotide sequence necessary to encode the polypeptide produced by anative Bt gene. For example, the native Bt cryLA(b) gene isapproximately 3.5 Kb in length and encodes a polypeptide which isapproximately 1150 amino acids in length. A full-length syntheticcryIA(b) Bt gene would be at least approximately 3.5 Kb in length.

Truncated Bt Genes: Refers to DNA sequences comprising less than thefull nucleotide sequence necessary to encode the polypeptide produced bya native Bt gene, but which encodes the active toxin portion of thepolypeptide. For example, a truncated synthetic Bt gene of approximately1.9 Kb encodes the active toxin portion of the polypeptide such that theprotein product exhibits insecticidal activity.

Tissue-preferred promoter: The term "tissue-preferred promoter" is usedto indicate that a given regulatory DNA sequence will promote a higherlevel of transcription of an associated structural gene or DNA codingsequence, or of expression of the product of the associated gene asindicated by any conventional RNA or protein assay, or that a given DNAsequence will demonstrate some differential effect; i.e., that thetranscription of the associated DNA sequences or the expression of agene product is greater in some tissue than in all other tissues of theplant.

"Tissue-specific promoter" is used to indicate that a given regulatoryDNA sequence will promote transcription of an associated coding DNAsequence essentially entirely in one or more tissues of a plant, or inone type of tissue, e.g. green tissue, while essentially notranscription of that associated coding DNA sequence will occur in allother tissues or types of tissues of the plant.

The present invention provides DNA sequences optimized for expression inplants, especially in maize plants. In a preferred embodiment of thepresent invention, the DNA sequences encode the production of aninsecticidal toxin, preferably a polypeptide sharing substantially theamino acid sequence of an insecticidal crystal protein toxin normallyproduced by Bacillus thuringiensis. The synthetic gene may encode atruncated or full-length insecticidal protein. Especially preferred aresynthetic DNA sequences which encode a polypeptide effective againstinsects of the order Lepidoptera and Coleoptera, and synthetic DNAsequences which encode a polypeptide having an amino acid sequenceessentially the same as one of the crystal protein toxins of Bacillusthuringiensis variety kurstaki, HD-1.

The present invention provides synthetic DNA sequences effective toyield high expression of active insecticidal proteins in plants,preferably maize protoplasts, plant cells and plants. The synthetic DNAsequences of the present invention have been modified to resemble amaize gene in terms of codon usage and G+C content. As a result of thesemodifications, the synthetic DNA sequences of the present invention donot contain the potential processing sites which are present in thenative gene. The resulting synthetic DNA sequences (synthetic Bt IPcoding sequences) and plant transformation vectors containing thissynthetic DNA sequence (synthetic Bt IP genes) result in surprisinglyincreased expression of the synthetic Bt IP gene, compared to the nativeBt IP gene, in terms of insecticidal protein production in plants,particularly maize. The high level of expression results in maize cellsand plants that exhibit resistance to lepidopteran insects, preferablyEuropean Corn Borer and Diatrea saccharalis, the Sugarcane Borer.

The synthetic DNA sequences of the present invention are designed toencode insecticidal proteins from Bacillus thuringiensis, but areoptimized for expression in maize in terms of G+C content and codonusage. For example, the maize codon usage table described in Murray etal., supra, is used to reverse translate the amino acid sequence of thetoxin produced by the Bacillus thuringiensis subsp. kurstaki HD-1cryIA(b) gene, using only the most preferred maize codons. The reversetranslated DNA sequence is referred to as the pure maize optimizedsequence and is shown as Sequence 4. This sequence is subsequentlymodified to eliminate unwanted restriction endonuclease sites, and tocreate desired restriction endonuclease sites. These modifications aredesigned to facilitate cloning of the gene without appreciably alteringthe codon usage or the maize optimized sequence. During the cloningprocedure, in order to facilitate cloning of the gene, othermodifications are made in a region that appears especially susceptibleto errors induced during cloning by the polymerase chain reaction (PCR).The final sequence of the maize optimized synthetic Bt IP gene is shownin Sequence 2. A comparison of the maize optimized synthetic Bt IP genewith the native kurstaki cryIA(b) Bt gene is shown in FIG. 1.

In a preferred embodiment of the present invention, the protein producedby the synthetic DNA sequence is effective against insects of the orderLepidoptera or Coleoptera. In a more preferred embodiment, thepolypeptide encoded by the synthetic DNA sequence consists essentiallyof the full-length or a truncated amino acid sequence of an insecticidalprotein normally produced by Bacillus thuringiensis var. kurstaki HD-1.In a particular embodiment, the synthetic DNA sequence encodes apolypeptide consisting essentially of a truncated amino acid sequence ofthe Bt CryIA(b) protein.

The insecticidal proteins of the invention are expressed in a plant inan amount sufficient to control insect pests, i.e. insect controllingamounts. It is recognized that the amount of expression of insecticidalprotein in a plant necessary to control insects may vary depending uponspecies of plant, type of insect, environmental factors and the like.Generally, the insect population will be kept below the economicthreshold which varies from plant to plant. For example, to controlEuropean corn borer in maize, the economic threshold is .5 eggmass/plantwhich translates to about 10 larvae/plant.

The methods of the invention are useful for controlling a wide varietyof insects including but not limited to rootworms, cutworms, armyworms,particularly fall and beet armyworms, wireworms, aphids, corn borers,particularly European corn borers, sugarcane borer, lesser corn stalkborer, Southwestern corn borer, etc.

In a preferred embodiment of the present invention, the synthetic codingDNA sequence optimized for expression in maize comprises a G+Cpercentage greater than that of the native cryIA(b) gene. It ispreferred that the G+C percentage be at least about 50 percent, and morepreferably at least about 60 percent. It is especially preferred thatthe G+C percent be about 64 percent.

In another preferred embodiment of the present invention, the syntheticcoding DNA sequence optimized for expression in maize comprises anucleotide sequence having at least about 90 percent homology with the"pure" maize optimized nucleotide sequence of the native Bacillusthuringiensis cryLA(b) protein, more preferably at least about 95percent homology, and most preferably at least about 98 percent.

Other preferred embodiments of the present invention include syntheticDNA sequences having essentially the DNA sequence of SEQ ID NO: 4, aswell as mutants or variants thereof; transformation vectors comprisingessentially the DNA sequence of SEQ ID NO: 4; and isolated DNA sequencesderived from the plasmids pCIB4406, pCIB4407, pCIB4413, pCIB4414,pCIB4416, pCIB4417, pCIB4418, pCIB4419, pCIB4420, pCIB4421, pCIB4423,pCIB4434, pCIB4429, pCIB4431, pCIB4433. Most preferred are isolated DNAsequences derived from the plasmids pCIB4418 and pCIB4420, pCIB4434,pCIB4429, pCIB4431, and pCIB4433.

In order to construct one of the maize optimized DNA sequences of thepresent invention, synthetic DNA oligonucleotides are made with anaverage length of about 80 nucleotides. These oligonucleotides aredesigned to hybridize to produce fragments comprising the variousquarters of the truncated toxin gene. The oligonucleotides for a givenquarter are hybridized and amplified using PCR. The quarters are thencloned and the cloned quarters are sequenced to find those containingthe desired sequences. In one instance, the fourth quarter, thehybridized oligonucleotides are cloned directly without PCRamplification. Once all clones of four quarters are found which containopen reading frames, an intact gene encoding the active insecticidalprotein is assembled. The assembled gene may then be tested forinsecticidal activity against any insect of interest including theEuropean Corn Borer (ECB) and the sugarcane borer. (Examples SA and SB,respectively). When a fully functional gene is obtained, it is againsequenced to confirm its primary structure. The fully functional gene isfound to give 100% mortality when bioassayed against ECB. The fullyfunctional gene is also modified for expression in maize.

The maize optimized gene is tested in a transient expression assay, e.g.a maize transient expression assay. The native Bt cryIA(b) codingsequence for the active insecticidal toxin is not expressed at adetectable level in a maize transient expression system. Thus, the levelof expression of the synthesized gene can be determined. By the presentmethods, expression of a protein in a transformed plant can be increasedat least about 100 fold to about 50,000 fold, more specifically at leastabout 1,000 fold to at least about 20,000 fold.

Increasing expression of an insecticial gene to an effective level doesnot require manipulation of a native gene along the entire sequence.Effective expression can be achieved by manipulating only a portion ofthe sequences necessary to obtain increased expression. A full-length,maize optimized CryIA(b) gene may be prepared which contains a proteinof the native CryIA(b) sequence. For example, FIG. 7 illustrates afull-length, maize optimized CryIA(b) gene which is a synthetic-nativehybrid. That is, about 2 kb of the gene (nucleotides 1-1938 SEQ ID NO:8) is maize optimized, i.e. synthetic. The remainder, C-terminalnucleotides 647-1155 SEQ ID NO: 8, are identical to the correspondingsequence native of the CryIA(b) gene. Construction of the illustratedgene is described in Example 6, below.

It is recognized that by using the methods described herein, a varietyof synthetic/native hybrids may be constructed and tested forexpression. The important aspect of hybrid construction is that theprotein is produced in sufficient amounts to control insect pests. Inthis manner, critical regions of the gene may be identified and suchregions synthesized using preferred codons. The synthetic sequences canbe linked with native sequences as demonstrated in the Examples below.Generally, N-terminal portions or processing sites can be synthesizedand substituted in the native coding sequence for enhanced expression inplants.

In another embodiment of the present invention, the maize optimizedgenes encoding cryIA(b) protein may be manipulated to render the encodedprotein more heat stable or temperature stable compared to the nativecryIA(b) protein. It has been shown that the cryIA(b) gene found inBacillus thuringiensis kurstaki HD-1 contains a 26 amino acid deletion,when compared with the cryIA(a) and cryIA(c) proteins, in the --COOHhalf of the protein. This deletion leads to a temperature-sensitivecryIA(b) protein. See M. Geiser, EP 0 440 581, entitled"Temperaturstabiles Bacillus thuringiensis-Toxin". Repair of thisdeletion with the corresponding region from the cryIA(a) or cryIA(c)protein improves the temperature stability of the repaired protein.Constructs of the full-length modified cryIA(b) synthetic gene aredesigned to insert sequences coding for the missing amino acids at theappropriate place in the sequence without altering the reading frame andwithout changing the rest of the protein sequence. The full-lengthsynthetic version of the gene is assembled by synthesizing a series ofdouble-stranded DNA cassettes, each approximately 300 bp in size, usingstandard techniques of DNA synthesis and enzymatic reactions. Therepaired gene is said to encode a "heat stable" or "temperature-stable"cryIA(b) protein, since it retains more biological activity than itsnative counterpart when exposed to high temperatures. Specific sequencesof maize optimized, heat stable cryIA(b) genes encoding temperaturestable proteins are set forth in FIGS. 9 (SEQ ID NO: 10), 11 (SEQ ID NO:12), 13 (SEQ ID NO: 14), and 15 (SEQ ID NO: 16), and are also describedin Example 7, below.

The present invention encompasses maize optimized coding sequencesencoding other polypeptides, including those of other Bacillusthuringiensis insecticidal polypeptides or insecticidal proteins fromother sources. For example, cryIB genes can be maize optimized, and thenstably introduced into plants, particularly maize. The sequence of amaize optimized cryIB gene constructed in accordance with the presentinvention is set forth in FIG. 6 (SEQ ID NO: 6).

Optimizing a Bt IP gene for expression in maize using the maizepreferred codon usage according to the present invention results in asignificant increase in the expression of the insecticidal gene. It isanticipated that other genes can be synthesized using plant codonpreferences to improve their expression in maize or other plants. Use ofmaize codon preference is a likely method of optimizing and maximizingexpression of foreign genes in maize. Such genes include genes used asselectable or scoreable markers in maize transformation, genes whichconfer herbicide resistance, genes which confer disease resistance, andother genes which confer insect resistance.

The synthetic cryIA(b) gene is also inserted into Agrobacterium vectorswhich are useful for transformation of a large variety of dicotyledenousplant species. (Example 44). Plants stably transformed with thesynthetic cryIA(b) Agrobacterium vectors exhibit insecticidal activity.

The native Bt cryIA(b) gene is quite A+T rich. The G+C content of thefull-length native Bt cryIA(b) gene is approximately 39%. The G+Ccontent of a truncated native Bt cryIA(b) gene of about 2 Kb in lengthis approximately 37%. In general, maize coding regions tend to bepredominantly G+C rich. The modifications made to the Bt cryIA(b) generesult in a synthetic IP coding region which has greater than 50% G+Ccontent, and has about 65% homology at the DNA level with the nativecryIA(b) gene. The protein encoded by this synthetic CryIA(b) gene is100% homologous with the native protein, and thus retains full functionin terms of insect activity. The truncated synthetic CryIA(b) IP gene isabout 2 Kb in length and the gene encodes the active toxin region of thenative Bt kurstaki CryIA(b) insecticidal protein. The length of theprotein encoded by the truncated synthetic CryIA(b) gene is 648 aminoacids.

The synthetic genes of the present invention are useful for enhancedexpression in transgenic plants, most preferably in transformed maize.The transgenic plants of the present invention may be used to expressthe insecticidal CryIA(b) protein at a high level, resulting inresistance to insect pests, preferably coleopteran or lepidopteraninsects, and most preferably European Corn Borer (ECB) and SugarcaneBorer.

In the present invention, the DNA coding sequence of the synthetic maizeoptimized gene may be under the control of regulatory elements such aspromoters which direct expression of the coding sequence. Suchregulatory elements, for example, include monocot or maize and othermonocot functional promoters to provide expression of the gene invarious parts of the maize plant. The regulatory element may beconstitutive. That is, it may promote continuous and stable expressionof the gene. Such promoters include but are not limited to the CaMV 35Spromoter; the CaMV 19S promoter; A. tumefaciens promoters such asoctopine synthase promoters, mannopine synthase promoters, nopalinesynthase promoters, or other opine synthase promoters; ubiquitinpromoters, actin promoters, histone promoters and tubulin promoters. Theregulatory element may be a tissue-preferential promoter, that is, itmay promote higher expression in some tissues of a plant than in others.Preferably, the tissue-preferential promoter may direct higherexpression of the synthetic gene in leaves, stems, roots and/or pollenthan in seed. The regulatory element may also be inducible, such as byheat stress, water stress, insect feeding or chemical induction, or maybe developmentally regulated. Numerous promoters whose expression areknown to vary in a tissue specific manner are known in the art. One suchexample is the maize phosphoenol pyruvate carboxylase (PEPC), which isgreen tissue-specific. See, for example, Hudspeth, R. L. and Grula, J.W., Plant Molecular Biology 12:579-589, 1989). Other greentissue-specific promoters include chlorophyll a/b binding proteinpromoters and RubisCO small subunit promoters.

The present invention also provides isolated and purified pith-preferredpromoters. Preferred pith-preferred promoters are isolated fromgraminaceous monocots such as sugarcane, rice, wheat, sorghum, barley,rye and maize; more preferred are those isolated from maize plants.

In a preferred embodiment, the pith-preferred promoter is isolated froma plant TrpA gene; in a most preferred embodiment, it is isolated from amaize TrpA gene. That is, the promoter in its native state isoperatively associated with a maize tryptophan synthase-alpha subunitgene (hereinafter "TrpA"). The encoded protein has a molecular mass ofabout 38 kD. Together with another alpha subunit and two beta subunits,TrpA forms a multimeric enzyme, tryptophan synthase. Each subunit canoperate separately, but they function more efficiently together. TrpAcatalyzes the conversion of indole glycerol phosphate to indole. Neitherthe maize TrpA gene nor the encoded protein had been isolated from anyplant before Applicants' invention. The Arabidopsis thaliana tryptophansynthase beta subunit gene has been cloned as described Wright et al.,The Plant Cell, 4:711-719 (1992). The instant maize TrpA gene has nohomology to the beta subunit encoding gene. The present invention alsoprovides purified pollen-specific promoters obtainable from a plantcalcium-dependent phosphate kinase (CDPK) gene. That is, in its nativestate, the promoter is operably linked to a plant CDPK gene. In apreferred embodiment, the promoter is isolated from a maize CDPK gene.By "pollen-specific," it is meant that the expression of an operativelyassociated structural gene of interest is substantially exclusively(i.e. essentially entirely) in the pollen of a plant, and is negligiblein all other plant parts. By "CDPK," it is meant a plant protein kinasewhich has a high affinity for calcium, but not calmodulin, and requirescalcium, but not calmodulin, for its catalytic activity.

To obtain tissue-preferred or tissue specific promoters, genes encodingtissue specific messenger RNA (mRNA) can be obtained by differentialscreening of a cDNA library. For example, a pith-preferred cDNA can beobtained by subjecting a pith cDNA library to differential screeningusing cDNA probes obtained from pith and seed mRNA. See, MolecularCloning, A Laboratory Manual, Sambrook et al. eds. Cold Spring HarborPress: New York (1989).

Alternately, tissue specific promoters may be obtained by obtainingtissue specific proteins, sequencing the N-terminus, synthesizingoligonucleotide probes and using the probes to screen a cDNA library.Such procedures are exemplified in the Experimental section for theisolation of a pollen specific promoter.

The scope of the present invention in regard to the pith-preferred andpollen-specific promoters encompasses functionally active fragments of afull-length promoter that also are able to direct pith-preferred orpollen-specific transcription, respectively, of associated structuralgenes. Functionally active fragments of a promoter DNA sequence may bederived from a promoter DNA sequence, by several art-recognizedprocedures, such as, for example, by cleaving the promoter DNA sequenceusing restriction enzymes, synthesizing in accordance with the sequenceof the promoter DNA sequence, or may be obtained through the use of PCRtechnology. See, e.g. Mullis et al., Meth. Enzymol. 155:335-350 (1987);Erlich (ed.), PCR Technology, Stockton Press (New York 1989).

Further included within the scope of the instant invention arepith-preferred and pollen-specific promoters "equivalent" to thefull-length promoters. That is, different nucleotides, or groups ofnucleotides may be modified, added or deleted in a manner that does notabolish promoter activity in accordance with known procedures.

A pith-preferred promoter obtained from a maize TrpA gene is shown inFIG. 24 (SEQ ID NO: 18). Those skilled in the art, with this sequenceinformation in hand, will recognize that pith-preferred promotersincluded within the scope of the present invention can be obtained fromother plants by probing pith libraries from these plants with probesderived from the maize TrpA structural gene. Probes designed fromsequences that are highly conserved among TrpA subunit genes of variousspecies, as discussed generally in Example 17, are preferred. Otherpollen-specific promoters, which in their native state are linked toplant CDPK genes other than maize, can be isolated in similar fashionusing probes derived from the conserved regions of the maize CDPK geneto probe pollen libraries.

In another embodiment of the present invention, the pith-preferred orpollen-specific promoter is operably linked to a DNA sequence, i.e.structural gene, encoding a protein of interest, to form a recombinantDNA molecule or chimeric gene. The phrase "operably linked to" has anart-recognized meaning; it may be used interchangeably with "operativelyassociated with," "linked to," or "fused to".

The structural gene may be homologous or heterologous with respect toorigin of the promoter and/or a target plant into which it istransformed. Regardless of relative origin, the associated DNA sequencewill be expressed in the transformed plant in accordance with theexpression properties of the promoter to which it is linked. Thus, thechoice of associated DNA sequence should flow from a desire to have thesequence expressed in this fashion. Examples of heterologous DNAsequences include those which encode insecticidal proteins, e.g.proteins or polypeptides toxic or inhibitory to insects or other plantparasitic arthropods, or plant pathogens such as fungi, bacteria andnematodes. These heterologous DNA sequences encode proteins such asmagainins, Zasloff, PNAS USA, 84:5449-5453 (1987); cecropins, Hultmarket al., Eur. J. Biochem. 127:207-217 (1982); attacins, Hultmark et al.,EMBO J. 2:571-576 (1983); melittin, gramicidin S, Katsu et al., Biochem.Biophys. Acta, 939:57-63 (1988); sodium channel proteins and syntheticfragments, Oiki et al. PNAS USA, 85:2395-2397 (1988); the alpha toxin ofStaphylococcus aureusm Tobkes et al., Biochem., 24:1915-1920 (1985);apolipoproteins and fragments thereof, Knott et al., Science 230:37(1985); Nakagawa et al., J. Am. Chem. Soc., 107:7087 (1985); alamethicinand a variety of synthetic amphipathic peptides, Kaiser et al., Ann.Rev. Biophys. Biophys. Chem. 16:561-581 (1987); lectins, Lis et al.,Ann. Rev. Biochem., 55:35-68 (1986); protease and amylase inhibitors;and insecticidal proteins from Bacillus thuringiensis, particularly thedelta-endotoxins from B. thuringiensis; and from other bacteria orfungi.

In a preferred embodiment of the invention, a pith-preferred promoterobtained from a maize TrpA subunit gene or pollen-specific promoterobtained from a maize CDPK gene is operably linked to a heterologous DNAsequence encoding a Bacillus thuringiensis ("B.t.") insecticidalprotein. These proteins and the corresponding structural genes are wellknown in the art. See, Hofte and Whiteley, Microbiol. Reviews,53:242-255 (1989).

While it is recognized that any promoter capable of directing expressioncan be utilized, it may be preferable to use heterologous promotersrather than the native promoter of the protein of interest. In thismanner, chimeric nucleotide sequences can be constructed which can bedetermined based on the plant to be transformed as well as the insectpest. For example, to control insect pests in maize, a monocot or maizepromoter can be operably linked to a Bt protein. The maize promoter canbe selected from tissue-preferred and tissue-specific promoters such aspith-preferred and pollen-specific promoters, respectively as disclosedherein.

In some instances, it may be preferred to transform the plant cell withmore than one chimeric gene construct. Thus, for example, a single plantcould be transformed with a pith-preferred promoter operably linked to aBt protein as well as a pollen-specific promoter operably linked to a Btprotein. The transformed plants would express Bt proteins in the plantpith and pollen and to a lesser extent the roots, outer sheath and braceroots.

For various other reasons, particularly management of potential insectresistance developing to plant expressed insecticidal proteins, it isbeneficial to express more than one insecticidal protein (IP) in thesame plant. One could express two different genes (such as two differentBacillus thuringiensis derived delta-endotoxins which bind differentreceptors in the target insect's midgut) in the same tissues, or one canselectively express the two toxins in different tissues of the sameplant using tissue specific promoters. Expressing two Bt genes (or anytwo insecticidal genes) in the same plant using three different tissuespecific promoters presents a problem for production of a plantexpressing the desired phenotype. Three different promoters driving twodifferent genes yields six different insecticidal genes that need to beintroduced into the plant at the same time. Also needed for thetransformation is a selectable marker to aid in identification oftransformed plants. This means introducing seven different genes intothe plant at the same time. It is most desired that all genes,especially the insecticidal genes, integrate into the plant genome atthe same locus so they will behave as a single gene trait and not as amultiple gene trait that will be harder to track during breeding ofcommercial hybrids. The total number of genes can be reduced by usingdifferential tissue specific expression of the different insecticidalproteins.

For example, by fusing cryIA(b) with the pollen and PEP carboxylasepromoters, one would obtain expression of this gene in green tissues andpollen. Fusing a pith-preferred promoter with the cryIB delta endotoxinfrom Bacillus thuringiensis would produce expression of thisinsecticidal protein most abundantly in the pith of a transformed plant,but not in seed tissues. Transformation of a plant with three genes, PEPcarboxylase/cryIA(b), pollen/cryIA(b), and pith/cryIB produces a plantexpressing two different Bt insecticidal endotoxins in different tissuesof the same plant. CryIA(b) would be expressed in the "outside" tissuesof a plant (particularly maize), that is, in those tissues whichEuropean corn borer feeds on first after hatching. Should ECB proveresistant to cryIA(b) and be able to burrow into the stalk of the plantafter feeding on leaf tissue and/or pollen, it would then encounter thecryIB delta-endotoxin and be exposed to a second insecticidal component.In this manner, one can differentially express two differentinsecticidal components in the same plant and decrease the total numberof genes necessary to introduce as a single genetic unit while at thesame time providing protection against development of resistance to asingle insecticidal component.

Likewise, a plant may be transformed with constructs encoding more thanone type of insecticidal protein to control various insects. Thus, anumber of variations may be constructed by one of skill in the art.

The recombinant DNA molecules of the invention may be prepared bymanipulating the various elements to place them in proper orientation.Thus, adapters or linkers may be employed to join the DNA fragments.Other manipulations may be performed to provide for convenientrestriction sites, removal of restriction sites or superfluous DNA.These manipulations can be performed by art-recognized methods. See,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, second edition, 1989. For example, methods suchas restriction, chewing back or filling in overhangs to provide bluntends, ligation of linkers, complementary ends of the DNA fragments canbe provided for joining and ligation. See, Sambrook et al., supra.

Other functional DNA sequences may be included in the recombinant DNAmolecule, depending upon the way in which the molecule is to beincorporated into the target plant genome. For instance, in the case ofAgrobacterium-mediated transformation, if Ti- or the Ri-plasmid is usedto transform the plant cells, the right and left borders of the T-DNA ofthe Ti- and Ri-plasmid will be joined as flanking regions to theexpression cassette. Agrobacterium tumefaciens-mediated transformationof plants has been described in Horsch et al., Science, 225:1229 (1985);Marton, Cell Culture Somatic Cell Genetics of Plants, 1:514-521 (1984);Hoekema, In: The Binary Plant Vector System Offset-Drukkerij KantersB.V., Alblasserdam, 1985, Chapter V Fraley, et al., Crit. Rev. PlantSci., 4:1-46; and An et al., EMBO J., 4:277-284 (1985).

The recombinant DNA molecules of the invention also can include a markergene to facilitate selection in recombinant plant cells. Examples ofmarkers include resistance to a biocide such as an antibiotic, e.g.kanamycin, hygromycin, chloramphenicol, paramomycin, methotrexate andbleomycin, or a herbicide such as imidazolones, sulfonylureas,glyphosate, phosphinothricin, or bialaphos. Marker genes are well knownin the art.

In another embodiment of the present invention, plants stablytransformed with a recombinant DNA molecule or chimeric gene asdescribed hereinabove are provided. The resultant transgenic plantcontains the transformed gene stably incorporated into its genome, andwill express the structural gene operably associated to the promoter inthe respective fashion.

Transgenic plants encompassed by the instant invention include bothmonocots and dicots. Representative examples include maize, tobacco,tomato, cotton, rape seed, soybean, wheat, rice, alfalfa, potato andsunflower. Preferred plants include maize, particularly inbred maizeplants.

All transformed plants encompassed by the instant invention may beprepared by several methods known in the art. A. tumefaciens-mediatedtransformation has been disclosed above. Other methods include directgene transfer into protoplasts, Paszkowski et al., EMBO J., 12:2717(1984); Loerz et al., Mol. Gen. & Genet., 1199:178 (1985); Fromm et al.,Nature 319:719 (1986); microprojectile bombardment, Klein et al.,Bio/Technology, 6:559-563 (1988); injection into protoplasts, culturedcells and tissues, Reich et al., Bio/Technology, 4:1001-1004 (1986); orinjection into meristematic tissues or seedlings and plants as describedby De La Pena et al., Nature, 325:274-276 (1987); Graves et al., PlantMol. Biol., 7:43-50 (1986); Hooykaas-Van Slogteren et al., Nature,311:763-764 (1984); Grimsley et al., Bio/Technology, 6:185 (1988); andGrimsley et al., Nature, 325:177 (1988); and electroporation,WO92/09696.

The expression pattern of a structural gene operatively associated withan instant tissue-preferred or tissue-specific promoter in a transformedplant containing the same is critical in the case where the structuralgene encodes an insecticidal protein. For example, the instantlydisclosed pith-preferred expression pattern will allow the transgenicplant to tolerate and withstand pathogens and herbivores that attackprimarily the pith, but also the brace roots, outer sheath and leaves ofthe plant since the protein will be expressed to a lesser extent butstill in an insect controlling amount in these plant parts, but yet inthe case of both types of promoters, will leave the seed of the plantunaffected.

EXAMPLES

The following examples further describe the materials and methods usedin carrying out the invention. They are offered by way of illustration,and not by way of limitation.

Example 1

General Methods

DNA manipulations were done using procedures that are standard in theart. These procedures can often be modified and/or substituted withoutsubstantively changing the result. Except where other references areidentified, most of these procedures are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, second edition, 1989.

Synthesis of DNA Oligomers:

DNA oligomers which are from about twenty to about ninety, preferablyfrom about sixty to about eighty nucleotides in length, are synthesizedusing an Applied Biosystems model 380B DNA synthesizer and standardprocedures. The oligomers are made using the updated SSCAF3 cycle on a0.2 μmole, wide pore, small scale ABI column. The end procedure is runtrityl off and the oligomer is cleaved from the column using the 380B'sautomatic cleavage cycle. The oligomers are then deblocked in excessammonium hydroxide (NH₄ OH) at 55° C. for 8-12 hours. The oligomers arethen dried in an evaporator using nitrogen gas. After completion, theoligomers are resuspended in 0.25-0.5 ml of deionized water.

Purification of Synthetic Oligomers:

An aliquot of each oligomer is mixed with an equal volume of bluedyeformamide mix with the final solution containing 0.05% bromophenolblue, 0.05% xylene cyanol FF, and 25% formamide. This mixture is heatedat 95° C. for 10 minutes to denature the oligomers. Samples are thenapplied to a 12% polyacrylamide-urea gel containing 7 M urea (Sambrooket al.). After electrophoresis at 300-400 volts for 34 hours using aVertical Slab Gel Unit (Hoefer Scientific Instruments, San Francisco,calif.), UV shadowing is used to locate the correct sized fragment inthe gel which was then excised using a razor blade. The purified gelfragment is minced and incubated in 0.4 M LiCl, 1 mM EDTA (pH 8) bufferovernight at 37° C.

Either of two methods is used to separate the oligomers from thepolyacrylamide gel remnants: Gene\X 25 μM porous polyethylene filterunits or Millipore's ultrafree-MC 0.45 μM filter units. The purifiedoligomers are ethanol precipitated, recovered by centrifuging in amicrofuge for 20 min at 4° C., and finally resuspended in TE (10 mMTris, 1 mM EDTA, pH 8.0). Concentrations are adjusted to 50 ng\|H 251based on absorption readings at 260 nm.

Kinasing Oligomers for Size Determinations:

To check the size of some of the oligomers on a sequencing gel, kinaselabeling reactions are carried out using purified synthetic oligomers ofeach representative size: 40 mers, 60 mers, 70 mers, 80 mers, and 90mers. In each 20 μl kinasing reaction, one pmole of purified oligomer isused in a buffer of 7.0 mM Tris pH 7.5, 10 mM KCl, 1 mM MgCl2), 0.5 mMDTT, 50 μg/ml BSA, 3000 μCi (3 pmoles) of 32P-gammaATP, and 8 units ofT4 polynucleotide kinase. The kinase reaction is incubated for 1 hour at37° C., followed by a phenol\chloroform extraction and three ethanolprecipitations with glycogen as carrier (Tracy, Prep. Biochem.11:251-268 (1981).

Two gel loadings (one containing 1000 cpm, the other containing 2000cpm) of each reaction are prepared with 25% formamide, 0.05% bromophenolblue, and 0.05% xylene cyanol FF. The kinased oligomers are boiled for 5minutes before loading on a 6% polyacrylamide, 7 M urea sequencing gel(BRL Gel Mix TM6, BRL, Gaithersburg, Md.). A sequencing reaction ofplasmid pUC18 is run on the same gel to provide size markers. Afterelectrophoresis, the gel is dried and exposed to diagnostic X-ray film(Kodak, X-OMAT AR). The resulting autoradiograph shows all purifiedoligomers tested to be of the correct size. Oligomers which had not beensized directly on the sequencing gel are run on a 6% polyacrylamide, 7 Murea gel (BRL Gel Mix TM6), using the sized oligomers as size markers.All oligomers are denatured first with 25% formamide at 100° C. for 5minutes before loading on the gel. Ethidium bromide staining of thepolyacrylamide gel allows all the oligomers to be visualized for sizedetermination.

Hybridizing Oligomers for Direct Cloning:

Oligomers to be hybridized are pooled together (from 1 μg to 20 μg totalDNA) and kinased at 37° C. for 1 hour in 1×Promega ligation buffercontaining 30 mM Tris-HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, and 1 mM dATP.One to 20 units of T4 polynucleotide kinase is used in the reaction,depending on the amount of total DNA present. The kinasing reactions arestopped by placing the reaction in a boiling water bath for fiveminutes. Oligomers to form the 5' termini of the hybridized moleculesare not kinased but are added to the kinased oligomers along withadditional hybridization buffer after heating. The pooled oligomers arein a volume of 50-100 ul with added hybridization buffer used to adjustthe final salt conditions to 100 mM NaCl, 120 mM Tris pH 7.5, and 10 mMMgCl2. The kinased and non-kinased oligomers are pooled together andheated in a boiling water bath for five minutes and allowed to slowlycool to room temperature over a period of about four hours. Thehybridized oligomers are then phenol\chloroform extracted, ethanolprecipitated, and resuspended in 17 μl of TE (10 mM Tris, 1 mM EDTA, pH8.0). Using this 17 μl, a ligation reaction with a final volume of 20 μlis assembled (final conditions=30 mM Tris-HCl pH 7.8, 10 mM MgCl2, 10 mMDTT, 1 mM ATP, and 3 units of T4 DNA ligase (Promega, Madison Wiss.).The ligation is allowed to incubate for about 2 hours at roomtemperature. The hybridized\ligated fragments are generally purified on2% Nusieve gels before andor after cutting with restriction enzymesprior to cloning into vectors. A 20 μl volume ligation reaction isassembled using 100 ng to 500 ng of each fragment with approximateequimolar amounts of DNA in 30 mM Tris-HCl pH 7.8, 10 mM MgCl2, 10 mMDTT, 1 mM ATP, and 3 units of T4 DNA ligase (Promega, Madison, Wiss.).Ligations are incubated at room temperature for 2 hours. After ligation,DNA is transformed into frozen competent E. coli cells using standardprocedures (Sambrook et al.) and transformants are selected on LB-agar(Sambrook et al.) containing 100 μg/ml ampicillin (see below).

PCR Reactions for Screening Clones in E. coli:

E. coli colonies which contain the correct DNA insert are identifiedusing PCR (see generally, Sandhu et al., BioTechniques 7:689-690(1989)). Using a toothpick, colonies are scraped from an overnight plateand added to a 20 μl to 45 μl PCR reaction mix containing about 50pmoles of each hybridizing primer (see example using primers MK23A28 andMK25A28 to select orientation of SacII fragment in pHYB2#6), 200 μm to400 mM of each dNTP, and 1×reaction buffer (Perkin Elmer Cetus, Norwalk,Conn.). After boiling the E. coli\PCR mix in a boiling water bath for 10minutes, 5 μl of Taq polymerase (0.5 units)(Perkin Elmer Cetus, Norwalk,Conn.) in 1×reaction buffer is added. The PCR reaction parameters aregenerally set with a denaturing step of 94° C. for 30 seconds, annealingat 55° C. for 45 seconds, and extension at 72° C. for 45 seconds for 30to 36 cycles. PCR reaction products are run on agarose or Nusieveagarose (FMC) gels to detect the correct fragment size amplified.

Ligations:

Restriction enzyme digested fragments are either purified in 1% LGT (lowgelling temperature agarose, FMC), 2% Nusieve (FMC), or 0.75% agaroseusing techniques standard in the art. DNA bands are visualized withethidium bromide and bands are recovered from gels by excision with arazor blade. Fragments isolated from LGT are ligated directly in theLGT. Ten microliters of each recovered DNA fragment is used to assemblethe ligation reactions, producing final ligation reaction volumes ofabout 23 μl. After excision with a razor blade, the recovered gel bandscontaining the desired DNA fragments are melted and brought to 1×ligasebuffer and 3 units of T4 DNA ligase (Promega) are added as describedabove. Fragments isolated from either regular agarose or Nusieve agaroseare purified from the agarose using ultrafree-MC 0.45 μM filter units(Millipore) and the fragments are ligated as described above. Ligationreactions are incubated at room temperature for two hours beforetransforming into frozen competent E. coli cells using standardprocedures (Sambrook et al.).

Transformations:

Frozen competent E. coli cells of the strain DH5alpha or HB101 areprepared and transformed using standard procedures (Sambrook et al.). E.Coli "SURE" competent cells are obtained from Stratagene (La Jolla,Calif.). For ligations carried out in LGT agarose, after ligationreactions are complete, 50 mM CaCl2 is added to a final volume of about150 μl and the solution heated at approximately 65° C. for about 10minutes to completely melt the agarose. The solution is then mixed andchilled on ice for about 10 minutes before the addition of about 200 μlof competent cells which had been thawed on ice. This mixture is allowedto incubate for 30 minutes on ice. The mixture is next heat shocked at42° C. for 60 seconds before chilling on ice for two minutes. Next, 800μl of SOC media (20% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mMKCl, adjusted to pH 8 with 5 N NaOH, 20 mM MgCl2:MgSO4 mix, and 20 mMglucose; Sambrook et al.) is added and the cells are incubated at 37° C.with shaking for about one hour before plating on selective mediaplates. Plates typically are L-agar (Sambrook et al.) containing 100μg/ml ampicillin.

When ligations are carried out in a solution without agarose, typically200 μl of frozen competent E. coli cells (strain DH5alpha (BRL,Gaithersburg, Md. or Sure cells, Stratagene, La Jolla, Calif.) arethawed on ice and 5 μl of the ligation mixture added. The reaction isincubated on ice for about 45 to 60 minutes, the cells are then heatshocked at 42° for about 90 seconds. After recovery at room temperaturefor about 10 minutes, 800 μl of SOC medium is added and the cells arethen incubated 1 hour at 37° C. with shaking and plated as above.

When screening for inserts into the beta-galactosidase gene in some ofthe standard vectors used, 200 μl of the recovered transformationmixture is plated on LB-agar plates containing 0.008% X-gal, 80 μM IPTG,and 100 μg/ml ampicillin (Sambrook et al.). The plates are incubated at37° overnight to allow selection and growth of transformants.

Miniscreening DNA:

Transformants from the selective media plates are grown and theirplasmid structure is examined and confirmed using standard plasmidmini-screen procedures (Sambrook et al.). Typically, the "boiling"procedure is used to produce small amounts of plasmid DNA for analysis(Sambrook et al.). Alternatively, an ammonium acetate procedure is usedin some cases. This procedure is a modification of that reported byShing-yi Lee et al., Biotechniques 9:676-679 (1990).

1) Inoculate a single bacterial colony from the overnight selectionplates into 5 ml (can be scaled down to 1 ml) of TB (Sambrook et al.)medium and grow in the presence of the appropriate antibiotic.

2) Incubate on a roller at 37° C. overnight.

3) Collect 5 ml of bacterial cells in a plastic Oakridge tube and spinfor 5 min. at 5000 rpm in a Sorvall SS-34 rotor at 4° C.

4) Remove the supernatant.

5) Resuspend the pellet in 1 ml of lysis buffer (50 mM glucose, 25 mMTris-HCl(pH 8.0), 10 mM EDTA and 5 mg/ml lysozyme), vortex for 5seconds, and incubate at room temperature for 5 min.

6) Add 2 ml of freshly prepared alkaline solution (0.2 N NaOH, 1% sodiumdodecyl sulfate), tightly secure lid, mix by inverting 5 times and placetube in an ice-water bath for 5 min.

7) Add 1.5 ml of ice-cold 7.5 M ammonium acetate (pH 7.6) to thesolution, mix by inverting the tube gently 5 times and place on anice-water bath for 5 min.

8) Centrifuge mixture at 9000 rpm for 10 min. at room temperature.

9) Transfer clear supernatant to a 15 ml Corex tube and add 0.6 volumesof isopropanol (approx. 2.5 ml). Let sit at room temperature for 10 min.

10) Centrifuge the mixture at 9000 rpm for 10 min. at room temperatureand discard the supernatant.

11) Resuspend the pellet in 300 ul of TE buffer. Add 6 ul of a stock ofRNase A & T1 (made as a 200 ul solution by adding 180 ul of RNase A(3254 Units/mg protein, 5.6 mg protein/ml) and 20 ul of RNase T1(481Units/ug protein, 1.2 mg protein/ml)). These stocks may be purchasedfrom USB(US Biochemical). Transfer to a microcentrifuge tube andincubate at 37° C. for 15 min.

12) Add 75 ul of distilled water and 100 ul of 7.5 M ammonium acetateand incubate in an ice-water bath for 10 min.

13) Centrifuge the mixture at 14,000 rpm for 10 min. in a Beckmanmicrofuge at 4° C.

14) Precipitate by adding 2.5 volumes of 100% EtOH (approx. 1 ml) andincubate in an ice-water bath for 10 min.

15) Spin at 14,000 rpm for 10 min. in a microfuge.

16) Wash pellet with 70% ethanol (using 0.5 ml-1 ml). Dry the pellet andresuspend in 100 μl of 1×New England Biolabs restriction enzyme Buffer 4(20 mM Tris-HCl(pH 7.9), 10 mM magnesium acetate, 50 mM potassiumacetate, 1 mM DTT). Measure concentration and check purity byspectrophotometry at absorbances 260 and 280 nm.

For a more rapid determination as to whether or not a particularbacterial colony harbored a recombinant plasmid, a PCR miniscreenprocedure is carried out using a modification of the method described by(Sandhu, G. S. et al., 1989, BioTechniques, 7:689-690). Briefly, thefollowing mixture is prepared:

100 μl primer mix above, 20 μM each primer,

100 μl dNTP mix (2.5 mM each)

100 μl 10×AmpliTaq buffer (Perkin-Elmer Cetus, 1×buffer=10 mM Tris-HClpH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin)

700 μl deionized water.

20 μl of the above mixture is put into a a 0.5 ml polyproplyene PCRtube. A transformed bacterial colony is picked with a toothpick andresuspended in the mixture. The tube is put in a boiling water bath for10 minutes and then cooled to room temperature before adding 5 ∥l of themix described below:

265 μl deionized water

30 μl 10×Amplitaq buffer (Perkin-Elmer Cetus, 1×buffer=10 mM Tris-HCl pH8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin)

7.5 μl Taq polymerase

The samples are overlaid with 50 μl of mineral oil and PCR is carriedout for 30 cycles using the following parameters:

denature: 94° for 1 min

anneal: 55° for 1 min

extend: 72° for 45 seconds.

After PCR amplification, 1 μl of loading dye (30% glycerol, 0.25%Bromophenol blue, 0.25% xylene cyanol) is added to the whole reactionand 20 μl of the mixture is loaded on a 2% Nusieve, 1% agarose gel tosee if there is a PCR product of the expected size.

This procedure is used as an initial screen. Minipreps are subsequentlycarried out to confirm the structure of the plasmid and its insert priorto sequencing.

Example 2

AMPLIFICATION AND ASSEMBLY OF EACH QUARTER

Cloning fragments of the synthetic Bt cryIA(b) gene:

The synthetic gene was designed to be cloned in four pieces, eachroughly one quarter of the gene. The oligomers for each quarter werepooled to either be assembled by PCR, hybridization, or a combination ofhybridization followed by PCR amplification as described elsewhere.Synthetic quarters were pieced together with overlapping restrictionsites Aat II, NcoI, and Apa I between the 1st and 2nd, 2nd and 3rd, and3rd and 4th quarters respectively.

Each quarter of the gene (representing about 500 bp) was assembled byhybridizing the appropriate oligomers and amplifying the desiredfragment using PCR primers specific for the ends of that quarter. Twodifferent sets of PCR reactions employing two sets of slightly differentprimers were used. The PCR products of the two reactions were designedto be identical except that in the first reaction there was anadditional AATT sequence at the 5' end of the coding region and in thesecond reaction there was an AGCT sequence at the 3' end of a givenquarter. When the products of the two reactions for a particular quarterwere mixed (after removing the polymerase, primers and incompleteproducts), denatured, and subsequently re-annealed, a certain ratio(theoretically 50%) of the annealed product should have non-homologousoverhanging ends. These ends were designed to correspond to the "stickyends" formed during restriction digestion with EcoRI at the 5' end andHind III at the 3' end of the molecule. The resulting molecules werephosphorylated, ligated into an EcoRI/HindIII digested and phosphatasedBluescript vector, and transformed into frozen competent E. coli strainDH5alpha. After selection, the E. coli colonies containing the desiredfragment are identified by restriction digest patterns of the DNA.Inserts representing parts of the synthetic gene are subsequentlypurified and sequenced using standard procedures. In all cases, clonesfrom multiple PCR reactions are generated and sequenced. The quartersare then joined together using the unique restriction sites at thejunctions to obtain the complete gene.

Cloned quarters are identified by mini-screen procedures and the genefragment sequenced. It is found that errors are frequently introducedinto the sequence, most probably during the PCR amplification steps. Tocorrect such errors in clones that contain only a few such errors,hybridized oligomers are used. Hybridized fragments are digested atrestriction enzyme recognition sites within the fragment and cloned toreplace the mutated region in the synthetic gene. Hybridized fragmentsrange from 90 bp in length (e.g. the region that replaces the fragmentbetween the Sac II sites in the 2nd quarter) to the about 350 bp 4thquarter fragment that replaces two PCR induced mutations in the 4thquarter of the gene.

Due to the high error rate of PCR, a plasmid is designed and constructedwhich allows the selection of a cloned gene fragment that contains anopen reading frame. This plasmid is designed in such a manner that if anopen reading frame is introduced into the cloning sites, the transformedbacteria could grow in the presence of kanamycin. The construction ofthis vector is described in detail below. This selection system greatlyexpedites the progress by allowing one to rapidly identify clones withopen reading frames without having to sequence a large number ofindependent clones. The synthetic quarters are assembled in variousplasmids, including BSSK (Stratagene; La Jolla, Calif.), pUC18 (Sambrooket al.), and the Km-expression vector. Other suitable plasmids,including pUC based plasmids, are known in the art and may also be used.Complete sequencing of cloned fragments, western blot analysis of clonedgene products, and insect bioassays using European corn borer as thetest insect verify that fully functional synthetic Bt cryIA(b) geneshave been obtained.

Construction of the Km-expression vector to select open reading frames:The Km-expression vector is designed to select for fragments of thesynthetic gene which contain open-reading frames. PCR oligomers aredesigned which allow the fusion of the NPTII gene from Tn5 starting atnucleotide 13 (Reiss et al., EMBO J. 3:3317-3322 (1984)) with pUC18 andintroduce useful restriction sites between the DNA segments. Thepolylinker region contains restriction sites to allow cloning varioussynthetic Bt IP fragments in-frame with the Km gene. The 88 bp 5'oligomer containing the polylinker region is purified on a 6%polyacrylamide gel as described above for the oligomer PAGEpurification. A PCR reaction is assembled with a 1 Kb Bgl II\Sma Itemplate fragment which contains the NPT II gene derived from Tn5. ThePCR reaction mix contains 100 ng of template with 100 pmols of oligomersKE72A28 and KE74A28 (see sequences below), 200 nM dNTP, and 2.5 Units ofTaq polymerase all in a 50 μl volume with an equal volume of mineral oiloverlaid. Sequences of the primers are:

KE74A28

5'-GCAGATCTGG ATCCATGCAC GCCGTGAAGG GCCCTTCTAG AAGGCCTATC GATAAAGAGCTCCCCGGGGA TGGATTGCAC GCAGGTTC-3' (SEQ ID NO: 29)

KE72A28

5'-GCGTTAACAT GTCGACTCAG AAGAACTCGT CAAGAAGGCG-3' (SEQ ID NO: 30)

The PCR parameters used are: 94° C. for 45 seconds (sec), 55° C. for 45sec, and 72° C. for 55 sec with the extension at step 3 for 3 sec for 20cycles. All PCR reactions are carried out in a Perkin-Elmer Cetusthermocycler. The amplified PCR product is 800 bp and contains thepolylinker region with a translational start site followed by uniquerestriction sites fused in-frame with the Km gene from base #13 runningthrough the translational terminator. pUC:KM74 is the Km-expressioncassette that was assembled from the 800 bp Bgl II\Sal I polylinker/Kmfragment cloned in the PUC18 vector. The lacZ promoter allows the Kmgene to be expressed in E. coli. pUC:KM74 derivatives has to first beplated on LB-agar plates containing 100 μg/ml ampicillin to selecttransformants which can subsequently be screened on LB-agar platescontaining 25 μg/ml kanamycin/IPTG. Synthetic Bt IP gene fragments areassembled from each quarter in the Km-cassette to verify cloning ofopen-reading-frame containing fragments pieces. The first ECB activesynthetic Bt IP gene fragment, pBt:Km#6, is a Bt IP gene that shows Kmresistance. This fragment is subsequently discovered to containmutations in the 3rd and 4th quarter which are later repaired.

Example 2A

SYNTHESIS AND CLONING OF THE FIRST QUARTER OF THE SYNTHETIC GENE (basepairs 1 to 550)

The following procedures are followed in order to clone the firstquarter of the synthetic DNA sequence encoding a synthetic Bt cryIA(b)gene. The same procedures are essentially followed for synthesis andcloning of the other quarters, except as noted for primers andrestriction sites.

Template for Quarter 1: Mixture of equal amounts of purified oligomersU1-U7 and L1 to L7

PCR Primers:

Forward:

P1 (a): 5'-GTCGACAAGG ATCCAACAAT GG-3' (SEQ ID NO: 31)

P1 (b): 5'-AATTGTCGAC AAGGATCCAA CAATGG-3' (SEQ ID NO: 32)

Reverse:

P2 (a): 5'-ACACGCTGAC GTCGCGCAGC ACG-3' (SEQ ID NO: 33)

P2 (b): 5'-AGCTACACGC TGACGTCGCG CAG-3' (SEQ ID NO: 34)

Primer pair A1: P1(b)+P2(a)

Primer pair A2: P1(a)+P2(b)

The PCR reaction containing the oligomers comprising the first quarterof the synthetic maize-optimized Bt IP gene is set up as follows:

200 ng oligo mix (all oligos for the quarter mixed in equal amountsbased on weight)

10 μl of primer mix (1:1 mix of each at 20 μM; primers are describedabove)

5 μl of 10×PCR buffer

PCR buffer used may be either

(a) 1×concentration=10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.0,2 mM MgSO4, and 0.1% Triton X-100), or

(b) 1×concentration=10 mM Tris-HCl pH 8.3, 50 mM KCl 1.5 mM MgCl2, 0.01%wt/vol gelatin.

Components are mixed, heated in a boiling water bath for 5 minutes, andincubated at 65° C. for 10 minutes.

Next, the following reagents are added:

8 μl of dNTPs mixture (final concentration in the reaction=0.2 mM each)

5 units polymerase.

The Final Reaction Volume is 50 Microliters.

Oligomers are then incubated for 3 min at 72° C. and then a PCR cycle isrun. The PCR reaction is run in a Perkin Elmer thermocycler on a stepcycle protocol as follows:

denaturation cycle: 94° for 1 minute

annealing cycle: 60° for 1 minute

extension cycle: 72° for 45 seconds (+3 sec per cycle)

number of cycles: 15

After the reaction is complete, 10 μl of the PCR reaction is loaded on a2% Nusieve-GTG (FMC), 1% agarose analytical gel to monitor the reaction.The remaining 40 μl is used to clone the gene fragments as describedbelow.

PCR Products

The termini of the double stranded PCR product corresponding to thevarious primer pairs are shown (only upper strand):A1 AATTGTCGAC(SEQ IDNO:35) GCGTGT (554 bp) first qtr.A2 GTCGAC GCGTGTAGCT(SEQ ID NO:36)(554bp) first qtr.

Hybridization

40 μl of each of the PCR reactions described above is purified using achromaspin 400 column (Clonetech, Palo Alto, Calif.) according tomanufacturers directions. Five μg of carrier DNA was added to thereactions before loading on the column. (This is done for most of thecloning. However, in some reactions the PCR reactions arephenol:chloroform extracted using standard procedures (Sambrook et al.)to remove the Taq polymerase and the PCR generated DNA is recovered fromthe aqueous phase using a standard ethanol precipitation procedure.) Thecarrier DNA does not elute with the PCR generated fragments. The A1 andA2 reaction counterparts for each quarter are mixed, heated in a boilingwater bath for 10 minutes and then incubated at 65° C. overnight. Thereactions are then removed from the 65° bath and ethanol precipitatedwith 1 μl (20 μg) of nuclease free glycogen (Tracy, Prep. Biochem.11:251-268 (1981) as carrier. The pellet is resuspended in 40 μl ofdeionized water.

Phosphorylation Reaction

The phosphorylation reaction is carried out as follows:

40 μl DNA

2.5 μl 20 mM ATP

0.5 μl 10×BSA/DTT (1×=5 mM DTT, 0.5 mg/ml BSA)

1.0 μl 10×polynucleotide kinase buffer (1×=70 mM Tris.HCl,

pH 7.6, 0.1 M KCl, 10 mM MgCl₂)

2.0 μl polynucleotide kinase (New England Biolabs, 20 units).

Incubation is for 2 hours at 37° C.

The reaction is then extracted one time with a 1:1 phenol:chloroformmixture, then once with chloroform and the aqueous phase ethanolprecipitated using standard procedures. The pellet is resuspended in 10μl of TE.

Restriction Digests

20 μg of Bluescript vector (BSSK+, Stratagene, La Jolla, Calif.)

10 μl 10×restriction buffer (1×=20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 100mM NaCl)

5 μl Eco RI (New England Biolabs) 100 units

5 μl Hind III (New England Biolabs) 100 units

Final reaction volume is 100 μl.

Incubation is for 3 hours at 37°.

When completed, the reaction is extracted with an equal volume of phenolsaturated with TE (10 mM Tris.HCl pH 8.0 and 10 mM EDTA). Aftercentrifugation, the aqueous phase was extracted with an equal volume of1:1 mixture of (TE saturated) phenol:chloroform (the "chloroform" ismixed in a ratio of 24:1 chloroform:isoamyl alcohol), and finally theaqueous phase from this extraction is extracted with an equal volume ofchloroform. The final aqueous phase is ethanol precipitated (by adding10 μl of 3 M sodium acetate and 250 ∥l of absolute ethanol, left at 4°for 10 min and centrifuged in a microfuge at maximum speed for 10minutes. The pellet is rinsed in 70% ethanol and dried at roomtemperature for 5-10 minutes and resuspended in 100 μl of 10 mM Tris.HCl(pH 8.3).

Phosphatase Reaction

Vector DNA is routinely treated with phosphatase to reduce the number ofcolonies obtained without an insert. Calf intestinal alkalinephosphatase is typically used (Sambrook et al.), but other phosphataseenzymes can also be used for this step.

Typical phosphatase reactions are set up as below:

90 μl of digested DNA described above

10 μl of 10×Calf intestinal alkaline phosphatase buffer (1×=50 mMTris-HCl (pH 8.3), 10 mM MgCl2, 1 mM ZnCl2, 10 mM spermidine)

1 μl (1 unit) of calf intestinal alkaline phosphatase (CIP, BoehringerMannheim, Indianapolis, Ind.)

Incubation is at 37° C. for 1 hour.

The DNA is then gel purified (on a 1% low gelling temperature (LGT)agarose gel) and the pellet resuspended in 50 μl TE. Afterelectrophoresis, the appropriate band is excised from the gel using arazor blade, melted at 65° for 5 minutes and diluted 1:1 with TE. Thissolution is extracted twice with phenol, once with the abovephenol:chloroform mixture, and once with chloroform. The final aqueousphase is ethanol precipitated and resuspended in TE buffer.

Ligation:

To ligate fragments of the synthetic gene into vectors, the followingconditions are typically used.

5 μl of phosphorylated insert DNA

2 μl of phosphatased Eco RI/Hind III digested Bluescript vector heatedat 65° for 5 minutes, then cooled

1 μl 10×ligase buffer (1×buffer=30 mM Tris.HCl (pH 7.8), 10 mM MgCl2, 10mM DTT, 1 mM ATP)

1 μl BSA (1 mg/ml)

1 μl ligase (3 units, Promega, Madison, Wis.)

Ligase reactions are typically incubated at 16° overnight or at roomtemperature for two hours.

Transformation:

Transformation of ligated DNA fragments into E. coli is performed usingstandard procedures (Sambrook et al.) as described above.

Identification of recombinants

White or light blue colonies resulting from overnight incubation oftransformation plates are selected. Plasmids in the transformants arecharacterized using standard mini-screen procedures (Sambrook et al.) oras described above. One of the three procedures listed below aretypically employed:

(1) boiling DNA miniprep method

(2) PCR miniscreen

(3) Ammonium acetate miniprep.

The restriction digest of recombinant plasmids believed to contain thefirst quarter is set up as follows:

(a) Bam HI/Aat II digest: 10 μl DNA+10 μl 1×New England Biolabsrestriction enzyme Buffer 4

0.5 μl Bam HI (10 units)

0.5 μl Aat II (5 units)

Incubation is for about 2 hours at 37° C.

Clones identified as having the desired restriction pattern are nextdigested with Pvu II and with Bgl II in separate reactions. Only cloneswith the desired restriction patterns with all three enzyme digestionsare carried further for sequencing.

Sequencing of cloned gene fragments:

Sequencing is performed using a modification of Sanger's dideoxy chaintermination method (Sambrook et al.) using double stranded DNA with theSequenase 2 kit (United States Biochemical Corp., Cleveland, Ohio). Inall, six first quarter clones are sequenced. Of the clones sequenced,only two clones designated pQA1 and pQA5 are found to contain only onedeletion each. These deletions are of one base pair each located atposition 452 in pQA1 and position 297 in pQA5.

Plasmid pQA1 is used with pP1-8 (as described below) to obtain a firstquarter with the expected sequence.

Example 2B

SYNTHESIS AND CLONING OF THE SECOND QUARTER (base pairs 531 to 1050)

Template: oligomers U8-U14 and L8-L14

PCR Primers:

forward:

P3 (a): 5'-GCTGCGCGAC GTCAGCGTGT TCGG-3' (SEQ ID NO: 37)

P3 (b): 5'-AATTGCTGCG CGACGTCAGC GTG-3' (SEQ ID NO: 38)

Reverse:

P4 (a): 5'-GGCGTTGCCC ATGGTGCCGT ACAGG-3' (SEQ ID NO: 39)

P4 (b): 5'-AGCTGGCGT TGCCCATGGT GCCG-3' (SEQ ID NO: 40)

Primer pair B1: P3(b)+P4(a)

Primer pair B2: P3(a)+P4(b)

PCR ProductsB1 AATTGCTGCG(SEQ ID NO:41) AACGCC (524 bp) secondquarterB2GCTGCG AACGCCAGCT(SEQ ID NO:42)(524 bp)

Hybridization, PCR amplification, spin column size fractionation, andcloning of this gene fragment in Bluescript digested with Eco RI/HindIII are performed as described above for the first quarter (Example 2A).The PCR product for this quarter is about 529 bp in size representingthe second quarter of the gene (nucleotides 531 to 1050). Transformationis into frozen competent E. coli cells (DH5alpha) using standardprocedures described above (Sambrook et al.)

Miniscreen of pQB clones:

Miniprep DNA is prepared as described above and digested with (a) AatII/Nco I, (b) Pvu II and (c) with Bgl I to confirm the structure insertin the vector before sequencing.

Sequencing is performed as described above using the dideoxy method ofSanger (Sambrook et al.).

A total of thirteen clones for this quarter are sequenced. The secondquarter consistently contains one or more deletions between position 884and 887. In most cases the G at position 884 is deleted.

Plasmid pQB5 had only one deletion at position 884. This region liesbetween two Sac II sites (positions 859 and 949). Correction of thisdeletion is described in Example 3.

Clones of the first half (1-1050 bp).

A fragment for cloning the first half (quarters 1 and 2) of thesynthetic Bt maize gene as a single DNA fragment is obtained byrestriction digestion of the product of a PCR reaction comprising thefirst quarter and the second quarter. Restriction endonuclease Aat II isused to cut the DNA (following phenol extraction and ethanolprecipitation) in a 20 μl reaction. 15 μl of each of the Aat II digestedquarters is mixed and ligated (in a 50 μl volume by adding 5 μl of10×ligase buffer, (1×=30 mM Tris-HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 1mM ATP) 14 μl of deionized water and 1 μl of T4 DNA ligase, 3 units,Promega, Madison, Wis.) at room temperature for 2 hr. The result is anabout 1 kb fragment as judged by electrophoresis on a 1% agarose gel runusing standard conditions (Sambrook et al.) Ten μl of the ligationproduct is amplified by PCR using conditions described previously exceptthat only 5 cycles were run.

Primer Pair: HA=P1(a)+P4(b)

Primer Pair: HB=P1(b)+P4(a)

The product of these reactions is cloned into Bluescript (Stratagene, LaJolla, Calif.) as described for the individual quarters. This procedureis only done once i.e., all insert DNA is obtained in a particularregion from a single PCR reaction.

Thirty-six colonies are miniscreened with Sal I digests and Pvu IIdigests. All except 4 contain an insert of approximately 1 kb in size ofwhich at least 20 contain the correct Pvu II digestion pattern. Eight ofthese clones are selected for sequence analysis. One of the clones,P1-8, has the desired sequence between the Eco NI site (396 bp) and theDra III site (640 bp). This clone is used to obtain a plasmid with thedesired sequence up to the Dra III site (640 bp) in the second quarterwith pQA1 (first quarter with a deletion at position 452 bp describedpreviously.)

Example 2C

CLONING AND SYNTHESIS OF THIRD QUARTER (base pairs 1021 to 1500)

Template: Oligos U15-U20 and L15-L21

PCR primers:

forward

P5 (a): 5'-TTCCCCCTGT ACGGCACCAT GGGCAACGCC GC-3' (SEQ ID NO: 43)

P5 (b): 5'-AATTGTACGG CACCATGGGC AAC-3' (SEQ ID NO: 44)

reverse

P6 (a): 5'-GAAGCCGGGG CCCTTCACCA CGCTGG-3' (SEQ ID NO: 45)

P6 (b): 5'-AGCTGAAGCC GGGGCCCTTC ACC-3' (SEQ ID NO: 46)

Primer pair C1: P5(b)+P6(a)

Primer pair C2: P5(a)+P6(b)

PCR Product:C1 AATTGTACGG(SEQ ID NO:47) GGCTTC (475 bp) 3d qtrC2TTCCCCTGTACGG(SEQ ID NO:48) GGCTTCAGCT(SEQ ID NO:49)(484 bp)3d qtr

PCR reactions, spin column recovery of the correct sized DNA fragment,and ligation into vectors are performed as described above (Example 2A)using a Bluescript vector cut with Eco RI and Hind III. Theapproximately 479 base pair PCR product represents the third quarter ofthe synthetic gene (NT 1021-1500).

Transformation into frozen competent E. coli strain DH5alpha cells,selection and identification of transformants, characterization oftransformants by mini-screen procedures, and sequencing of the syntheticgene fragment in the vector are all as described above.

Mini screen of pQC clones:

The third quarter is miniscreened using standard procedures (Sambrook etal.). Miniprep DNA is cut with (a) Nco I/Apa I and (b) with Pvu II.Clones containing the correct restriction digest patterns are sequencedusing standard procedures. A total of 22 clones of the third quarter aresequenced. Three major deletion "hotspots" in the third quarter areidentified (a) at position 1083 (b) between position 1290-1397 and (c)between positions 1356-1362. In all clones except one, pQC8, there isalso consistently an insertion of a C at position 1365. In addition tothese mutations, the third quarter clones contain a large number ofother apparently random deletions. The common factor to the threemutational "hotspots" in the third quarter and the one in the secondquarter is that these regions are all flanked on either side bysequences that are about 80% C+G. Other regions containing 5 to 9 C-Gsin a row are not affected. The oligomers in U15, U16, U18, U19, L15,L16, L18 and L19 are redesigned to reduce the C+G content in theseregions. Five clones each from PCR reaction using the modified oligomersare sequenced.

Plasmid pQCN103 has the correct sequence for the third quarter exceptfor a change at position 1326. This change, which substitutes a G for aC, results in the substitution of one amino acid (leucine) for theoriginal (phenylalanine).

Example 2D

SYNTHESIS AND CLONING OF FOURTH QUARTER (base pairs 1480 to 1960)

The fourth quarter of the gene is obtained from a clone which isoriginally designed to comprise the third and fourth quarters of thegene. The "second half" of the synthetic gene is obtained from PCRreactions to fuse the third and fourth quarters. These reactions are runwith PCR primers P5(a) and P6(a) described above for the third quarterand primers P7(a) and P8(a) (described below). The reverse primer ismodified to include a Sac I site and a termination codon. Separatereactions for each quarter are run for 30 cycles using the conditionsdescribed above. The two quarters are joined together by overlapping PCRand subsequently digested with restriction enzymes Nco I and Sac I. Theresulting 953 bp fragment is cloned directionally into pCIB3054, whichhas been cut with Nco I/Sac I and treated with alkaline phosphatase.

pCIB3054 is constructed by inserting intron #9 of PEPcarboxylase (PEPCivs #9) in the unique Hpa I site of pCIB246 (described in detail inExample 4) pCIB246 is cut with HpaI and phosphatased with CIP usingstandard procedures described in Example 2A. PEPC ivs #9 is obtained byPCR using pPEP-10 as the template. pPEP-10 is a genomic subclonecontaining the entire maize PEP carboxylase gene encoding the C₄photosynthetic enzyme, plus about 2.2 Kb of 5'-flanking and 1.8 Kb of3'-flanking DNA. The 10 Kb DNA is ligated in the HindIII site of pUC18.(Hudspeth et al., Plant Molecular Biology, 12: 576-589 (1989). Theforward PCR primer used to obtain the PEPCivs#9 is GTACAAAAACCAGCAACTC(SEQ ID NO: 50) and the reverse primer is CTGCACAAAGTGGAGTAGT (SEQ IDNO: 51). The PCR product is a 108 bp fragment containing only thePEPcarboxylase intron #9 sequences. The PCR reaction is extracted withphenol and chloroform, ethanol precipitated phosphorylated withpolynucleotide kinase and treated with T4 polymerase to fill in the 3'nontemplated base addition found in PCR products (Clark, J. M., NucleicAcid Research, 16: 9677-9686 (1988)) using standard procedures.

The kinased fragment is blunt-end cloned into the HpaI site of pCIB246,using standard

procedures described earlier.

Amplification and Assembly of the Fourth Quarter

Template: U21 -U26 and L22-L28

PCR primers

FORWARD

P7 (a): 5'-TGGTGAAGGG CCCCGGCTTC ACCGG-3' (SEQ ID NO: 52)

REVERSE

P8 (a): 5'-ATCATCGATG AGCTCCTACA CCTGATCGAT GTGGTA-3' (SEQ ID NO: 53)

PRIMER PAIR 4: P7(a)+P8(a)

PRIMER PAIR 3: P5(A)+P6(a)

Primer pair for overlapping PCR: P7(a)+P8(a)

PCR Product

                          (SEQ ID NO:54)                                          fourth quarter:GGTGAA          ATCAGGAGCTCATCGATGAT                       

(484 bp) third quarter: TTCCCCCTGTA (SEQ ID NO:55)--------------TTCACCGG

(484 bp) second half: GGTGAA-------CATGATGAT (953 bp)

Four positive clones are identified by plasmid miniscreen and aresubsequently sequenced using standard procedures.

Plasmid Bt.P2 #1 contains approximately the correct fourth quartersequence except for two mutations. These mutations are at position 1523(substituting an A for a G, resulting in an amino acid change whichsubstitutes a His for an Arg) and at position 1634 (substituting a T fora C, resulting in an amino acid substitution of a Ser for a Thr).

Plasmid Bt.P2 #1 is used in the construction of pCIB4414 describedbelow. (The mistakes are ultimately corrected by hybridizing all theoligos of the fourth quarter, digesting with Apa I/Bst E II andreplacing that region in pCIB4414. Therefore, only sequences fromposition 1842-1960 remain of Bt.P2#1 in the final construct.)

Example 3

ASSEMBLY AND REPAIR OF THE FINAL SYNTHETIC GENE

The synthetic maize optimized Bt cryIA(b) gene is designed to be clonedin quarters. Using the PCR technique, however, results in mutations,which in most cases are deletions resulting in frameshift mutations.Plasmids containing individual quarters are therefore sequenced and thecorrect parts ligated together using standard procedures.

After obtaining first and second quarter clones with almost the desiredsequence, plasmids pEB 1Q#4 and pEB 1Q#5 are constructed to obtain thedesired sequence of the synthetic Bt gene up to the Dra III site at thebase pair position 634 (this mutation destroys the Dra III site). ThepEB1Q constructs are made by ligating a 3.9 Kb Eco NI\Bam HI fragmentfrom pP1-8 with a 400 bp fragment from pQA1. pEB 1Q#5 has the desiredsequence up to the Dra III site, but pEB 1Q#4 has a mutation at basepair position 378.

Plasmids p1H1M4 and p1H1M5 are constructed to repair the Dra III site inpEB1Q#4 and pEB1Q#5. Plasmids p1H1M#4 and #5 are made by ligating a 3.5Kb Nco I\Aat II fragment from pEB1Q#4 and #5 respectively, with a 500 bpNco I\Aat II fragment from pQB5. Plasmid p1H1M5 contains a mutationbetween the Sac II sites at position 884 in the second quarter of thesynthetic Bt gene. Plasmid p1H1M4 contains the additional mutation asdescribed in its precursor construct pEB1Q#4.

The Sac II site in the Bluescript vector region of p1H1M4 is deleted bycutting p1H1M4 with Not I and Sac I and converting these sites to bluntends using T4 DNA polymerase under standard conditions before ligatingthis 3.9 Kb fragment to make p1H1M4 S. Deleting the Sac II site in thevector region allows the 90 bp Sac II fragment with the mutation atposition 884 in the 2nd quarter of p1H1M4 S to be removed prior toreplacement with a 90 bp Sac II fragment. Oligomers U\L 12 and 13 arekinased and hybridized (described above) before cutting with Sac II andisolating a 90 bp fragment on a 2% Nusieve gel. The Sac II fragment isligated into the about 3.8 Kb Sac II cut p1H1M4 S vector which has beenphosphatased with CIP. The repaired Sac II construct is called pHYB2#6.The orientation of the Sac II fragment in pHYB2#6 is detected by PCRscreening as described earlier using the following primers:

MK23A28=5'-GGGGCTGCGGATGCTGCCCT-3' (SEQ ID NO: 56)

MK25A28=5'-GAGCTGACCCTGACCGTGCT-3' (SEQ ID NO: 57)

MK26A28=5'-CACCTGATGGACATCCTGAA-3' (SEQ ID NO: 58)

Running the PCR reactions with 50 pmoles of primers MK23A28 and MK25A28produces an approximate 180 bp fragment, indicating the insertedfragment bounded by the Sac II sites in pHYB2#6 is in the correctorientation. Using primers MK25A28 and MK26A28 in the PCR screening actsas the negative control producing an approximate 180 bp fragment only inconstructs containing the Sac II bounded fragment in the wrongorientation. pHYB2#6 sequence is determined using standard procedures.

pHYB2#6 has one mutation at position 378 which needed to be repaired toobtain a first quarter containing the desired sequence.

Plasmid p1HG#6 contains the desired sequence for the entire first halfof the synthetic Bt gene. p1HG#6 is made from a 3.4 Kb Aat II\Nco Ifragment of p1H1M5#2 ligated to a 500 bp Aat II\Nco I fragment frompHYB2#6.

To identify clones or partial clones of the synthetic gene which containopen reading frames, the kanamycin selection vector (described above) isused. The fourth quarter of the synthetic Bt gene is the first put intothe kanamycin cassette. pKM74-4 contains the approximately 500 bp ApaI\Cla I fragment from plasmid BtP2 (which had been previouslytransformed into a dam- E. coli strain (PO-100) to be able to cut withCla I), ligated to pUC:KM74 cut with Apa I\Cla I. Plasmid pKM74-4displays kanamycin resistance but is later found to contain twosubstitution mutations at positions 1523 and 1634 (mutations aredescribed above in the section on cloning the fourth quarter; they aresubstitutions, not deletions or insertions). The correct first half ofthe synthetic Bt gene from plasmid p1HG#6 is inserted into plasmidpKM74-4. The resulting plasmid, called pKm124, is made from the about3.9 Kb Apa I\Bam HI fragment derived from pKM74-4 ligated to 1 Kb ApaI\Bam HI fragment from p1HG#6. pKm124 shows kanamycin resistance. Thisplasmid contains the first, second, and fourth quarters of the syntheticgene forming a single open reading frame.

The third quarter of the synthetic gene is next cloned into pKM124. Thefirst functional clone, in plasmid pBt:Km#6, is a functional copy of thetruncated synthetic cryIA(b) gene in the Km-cassette which displayskanamycin resistance but which contains deletion mutations between thethird and fourth quarters. Plasmid pBt:Km#6 is made from theapproximately 5 Kb Apa I\Nco I pKM124 vector fragment ligated to theapproximately 500 bp Apa I\Nco I fragment from pQCN103 (pQCN103 containsa mismatch mutation at position 1326 which is repaired later).Contaminating nuclease activity appears to have deleted the Apa I sitebetween the third and fourth quarters in pBt:Km#6. The Bt gene encodedby the synthetic gene in plasmid pBT:Km#6 has about 50-60% of the nativeproteins' activity against ECB. The 2 Kb Sma I\Bam HI fragment frompBt:Km#6 is inserted into a 35S:expression cassette to make a plasmidcalled 35S:Bt6.

Two functional synthetic Bt clones, each with mutations, are initiallyobtained: plasmids pBT:KM#6 and pCIB4414. pCIB4414, which is 100% activein insect bioassays against European corn borer compared with the nativegene, contains substitution mutations in the third and fourth quartersat positions 1323, 1523, and 1634.

pCIB4414 is constructed from two plasmids, MG3.G4#18 and 1HG which isdescribed above. MG3.G4#18 is obtained by cloning the Apa I/Kpn Ifragment in plasmid Bt.P2#1 into pQCN103 (using those same restrictionsites). This produces a plasmid containing the third and fourth quartersof the gene. The first half of the synthetic gene from plasmid 1HG iscut with Bam HI and Nco I and moved into MG3.G4#18 (containing the thirdand fourth quarters of the gene). The resulting plasmid, pCIB4414,contains a functional version of the synthetic gene. While beingfunctional, the synthetic gene in this plasmid contains three errors;position 1326 (G substituted for a C), position 1523 (substitute A for aG), and at position 1634 (substitution of a T for a C).

The fourth quarter in pCIB4414 is replaced with a 354 bp fourth quarterApa I\Bst E II fragment obtained from hybridizing, ligating, andrestriction cleaving fourth quarter oligomers as described earlier, andisolating the fragment from a 2% Nusieve agarose gel. pCIB4408 is asynthetic Bt gene clone obtained by replacing the fourth quarterfragment in pCIB4414 with the hybridized fourth quarter fragment. Toinsert the CaMV 35S promoter in front of the synthetic Bt gene, pCIB4406is made from a 4 Kb Eco N\Kpn I fragment from plasmid p35SBt6 and 1.8 KbEco N\Kpn I fragment from pCIB4408.

pCIB4406 is 100% active (as compared with the protein from the nativegene) against ECB but contains the substitution mutation in the thirdquarter of the synthetic gene at position 1323 resulting in an aminoacid substitution of a leucine for a phenylalanine. Plasmid pBS 123#13is used to repair this mutation.

The third quarter fragment in plasmid pBS 123#13 is made from anapproximately 479 bp hybridized oligomer generated fragment. Thirdquarter oligomers U15-U20 and L15-L21 are kinased, hybridized, andligated as described above. PCR reactions are carried out as describedabove with primers P5(a) and P6(b) for 15 cycles. The PCR product istreated with proteinase K at a final concentration of about 50 μg\ml inan approximate 95 μl volume for 30 minutes at 37° C. followed by 10minutes at 65° C. (Crowe et al., Nucleic Acid Research 19:184, 1991.)Subsequently, the product is phenol\chloroform extracted and ethanolprecipitated using standard procedures before cutting with restrictionenzymes Apa I and Nco I.

The approximate 450 bp Apa I\Nco I PCR fragment is ligated to the 3.8 KbApa I\Nco I vector fragment from p1HG#6 to make pBS123#13. PlasmidpBS123#13 contains the desired sequence for the third quarter of themaize optimized cryIA(b) gene from position 1319 at the Nsp I sitethrough the Apa I site at position 1493. This 170 bp Nsp I\Apa Ifragment from pBS 123#13 is used in the fully active synthetic cryIA(b)gene in plasmid pCIB4418.

Western Blot Analysis:

Western blot analyses of various transformants are performed using crudeextracts obtained from E. coli grown on selective plates. Using atoothpick, cultures are scraped from fresh plates containing thetransformants of interest which have been grown overnight at 37° C. Thepositive control for expression of the Bt gene in E. coli was aconstruct called pCIB3069 which contains the native Bt-k gene fused withthe plant expressible CaMV 35S promoter. pCIB3069 also contains the 35Spromoter operably linked to the hygromycin resistance gene, 35Spromoter, with Adh intron #1 operably linked to the GUS gene, and 35Spromoter operably linked to a gene coding for the production of thenative Bt cryIA(b) IP. A negative control of E. coli which does notcontain a Bt gene is also included in the analyses. Cultures areresuspended in 100 μl of loading buffer containing 62 mM Tris-HCl pH6.8, 1% SDS, 0.0025% bromophenol blue, 10% glycerol and 7.5%mercaptoethanol. After heating the mixtures at 95° C. for 10 minutes,the preparations are sonicated for 1-3 seconds. The debris iscentrifuged in a microfuge at room temperature for about 5 minutes and10 to 15 μl of each sample is loaded onto an acrylamide gel with a 10%running gel below a 6% stacking gel (Laemmli, Nature 227;680-685(1970)).After electrophoresis overnight at 10 mAmps, proteins are transferredfrom the gel to an Immobilon membrane (Millipore). The transfer is doneusing an electrophoretic Blotting Unit (American BioNuclear, Emeryville,Calif.) in transfer buffer (20 mM Tris, 150 mM glycine, and 20%methanol) for 1.5 hours at 450 mAmps.

Buffers for western blotting included:

    ______________________________________                                        Blocking buffer:  2% Tween-20                                                                   30 mM Tris-HCl pH 10.2                                                        150 nM NaCl                                                 Wash buffer:      0.05% Tween-20                                                                30 mM Tris-HCl pH 10.2                                                        150 mM NaCl                                                 Developing buffer:                                                                              100 mM Tris-HCl pH 9.6                                                        100 mM NaCl                                                                   10 mM MgCl2                                                 ______________________________________                                    

After transfer is complete, the membrane is incubated for about tenminutes in the blocking buffer. Three 15 minute washes with wash bufferare done before the first antibody treatment. The first antibody is animmunoaffinity purified rabbit or goat antibody prepared using theCryIA(b) protein as the antigen (Ciba-Geigy, RTP, N.C.; Rockland Inc.,Gilbertsville, Pa.; and Berkeley Antibody CO., Richmond, Calif.). ThecryIA(b) specific antibody is treated immediately before use with E.coli lysate from Bio-Rad in a 1 ml volume with 5 μg of antibody, 50 μlof E. coli lysate in the wash buffer solution. This mixture is incubatedfor 1 hour at room temperature before diluting it 1 to 30 for a finaldilution of 1:6000 with wash buffer. Incubation of the membrane with thefirst antibody is at room temperature for 1.5 hours.

Three 10 minute washes are done between the 1st and 2nd antibodytreatments. The second antibody is either rabbit anti-goat or goatanti-rabbit/alkaline phosphatase conjugate (Sigma, St. Louis, Mo.).Incubation with the alkaline phosphatase conjugate is carried out atroom temperature for one hour using a 1 to 6000 dilution in wash buffer.Six 10 minute washes are done between the second antibody treatment anddeveloping the western blot. The western blot is developed in 100 ml ofdeveloping buffer with 440 μl of nitroblue tetrazolium in 70% dimethylformamide (75 mg\ml), and 330 μl of 5-bromo-4-chloro-indolyl-phosphatein 100% dimethyl formamide (50 mg\ml). After developing for 15 to 30minutes, the membrane is washed in water and air dried.

Example 4

CONSTRUCTION OF TRANSFORMATION VECTORS

Construction of pCIB710 and derivatives.

CaMV 35S Promoter Cassette Plasmids pCIB709 and pCIB710 are constructedas shown in Rothstein et al., Gene 53:153-161 (1987). pCIB710 containsCaMV promoter and transcription termination sequences for the 35S RNAtranscript (Covey et al., Nucl. Acids. Res., 9:6735-6747 (1981)). A 1149bp BglII restriction fragment of CaMV DNA (bp 6494-7643 in Hohn et al.,Current Topics in Microbiology and Immunology, 96:194-220 and AppendicesA to G (1982)) is isolated from CaMV DNA by preparative agarose gelelectrophoresis as described earlier The fragment is mixed withBamHI-cleaved plasmid pUC19 DNA, treated with T4 DNA ligase, andtransformed into E. coli. (Note the BamHI restriction site in theresulting plasmid is destroyed by ligation of the BglII cohesive ends tothe BamHI cohesive ends.)

The resulting plasmid, called pUC19/35S, is then used inoligonucleotide-directed in-vitro mutagenesis to insert the BamHIrecognition sequence GGATCC immediately following CaMV nucleotide 7483in the Hohn reference. The resulting plasmid, pCIB710, contains the CaMV35S promoter region and transcription termination region separated by aBamHI restriction site. DNA sequences inserted into this BamHI site willbe expressed in plants by these CaMV transcription regulation sequences.(Also note that pCIB710 does not contain any ATG translation initiationcodons between the start of transcription and the BamHI site). pCIB710is modified to produce pCIB709 by inserting a Bam HI fragment containingthe coding sequence for hygromycin phosphotransferase from pLG90(Rothstein et al., Gene, 53:153-161 (1987)) in the Bam HI site.

pCIB709 is modified to produce pCIB996 by removing the ATG just upstreamfrom the initiation codon of the hygromycin phosphotranserase gene usingstandard mutagenesis techniques while inserting a Bgl II restrictionsite at this location. The resulting plasmid, pCIB996, is furthermodified to remove the Bam HI, Sma I and Bgl II sites in the 5'untranslated leader region located 5' of the initiation codon for theinitiation codon. The result is a change of DNA base sequence from-TATAAGGATC CCGGGGGCA AGATCTGAGA TATG (SEQ ID NO: 59)-Hyg to -TATAAGGATCTGAGATATG (SEQ ID NO: 59 with nucleotides 11-24 deleted)-Hyg. Theresulting plasmid is known as pCIB3073.

Alternatively, pCIB710 is modified to produce pCIB900, by inserting theBam HI--Bcl I fragment of pCIB 10/35SBt, which contains the 645 aminoacid Bt coding sequence, described in Part C4 below, into the Bam HIsite of pCIB710 to create pCIB710/35SBt. To introduce an antibioticresistance marker, pCIB709 is cut with Sal I, a Kpn I/Sal I adaptor isligated and the resulting ligation product is cut with Kpn I. The Kpnfragment of pCIB709 containing the 35S/hygromycin resistance gene isinserted into the Kpn I site of pCIB710/35SBt to produce pCIB900.

Genes useful as the selectable marker gene include the hygromycinresistance gene described in Rothstein et al., Gene 53: 153-161 (1987).The hygromycin gene described in this reference is moved into a pUCplasmid such as pCIB710 or pCIB709 and the "extra" ATG upstream from thehygromycin phosphotransferase coding sequence is removed to createpCIB996. This modified pCIB996 gene is further modified to remove aBglII, BamfHI and SmaI sites from the 5' region of the gene usingstandard techniques of molecular biology to make pCIB3073.

pCIB932 is a pUC19-based plasmid containing the chimeric genePep-C:promoter\Bt\Pep-C:terminator. It is composed of fragments derivedfrom pPEP-10, a HindIII subclone of a genomic clone, H1-lambda-14, PNASUSA, 83:2884-2888 (1986), of the maize gene encoding the PEP carboxylaseenzyme active in photosynthesis, and from pCIB930, which is a BamHIfragment containing the 645 amino acid truncated form of the the cryIAbendotoxin gene in the BamHI site of pUC18.

The 2.6 kb EcoRI-XhoI fragment from pPEP-10, containing the polyAaddition site from the PEP carboxylase gene, is isolated and digestedwith PstI and HincII. The restriction digest is ligated with PstI/HincIIdigested pUC18, transformed into E. coli and transformants screened forthose containing a 412 bp PstI-HincII insert in pUC18 and the insertverified by sequencing. The resulting plasmid is called pCIB931.

The nuclear gene encoding the phosphoenolpyruvate carboxylase isozyme("Pep-C") is described in Hudspeth et al., Plant Molecular Biology, 12:579-589 (1989). pCIB932 is constructed by the ligation of threefragments. The first fragment, containing the PEP-C transcriptionterminator, is produced by digesting pCIB931 to completion with HindIII,partially with SphI and the 3098 bp fragment isolated. The secondfragment, containing the Bt endotoxin coding sequence, is produced bydigesting pCIB930 with NcoI and SphI and isolating the 1950 bp fragment.The third fragment, containing the PEP-C promoter, is produced bydigesting pPEP-10 to completion with HindIII, partially with NcoI andisolating the 2.3 kb fragment. The ligation mix is transformed into E.coli, transformants with the correct insertion identified and the insertverified by sequencing.

pCIB932 is cut with PvuII to generate a 4.9 Kb fragment containing themaize Pep-C:promoter\Bt\Pep-C:terminator and purified on a 1% LGTagarose gel in 1×TAE. The linearized pCIB3079 vector and the 4.9 Kbinsert from pCIB932 are ligated using T4 DNA ligase in LGT to makepCIB4401. pCIB4401 is a maize transformation vector containing thechimeric genes: 35S:promoter\PAT\35S:terminator,Pep-C:promoter\Bt\Pep-C: terminator, and 35S:promoter\AdhI #1intron\GUS\35S: terminator.

Construction of pCIB246 (35S-GUS-35S)

A CaMV 35S promoter cassette, pCIB246, is constructed as follows.

The DdeI restriction site at nucleotide position 7482 of the CaMV genome(Franck et al., Cell, 21:285-294 (1980)) is modified by insertion of a48 bp oligonucleotide containing several restriction enzyme sitesincluding an NcoI (CCATGG) site, a SalI (GTCGAC) site, and an SstI(GAGCTC) site. This altered CaMV 35S promoter is inserted into a pUC19vector that had been modified to destroy the vector's SstI and SalIsites. Thus, the CaMV 35S promoter of pCIB 1500 contains unique SstI andSalI sites for cloning.

pCIB 1500 is digested with SstI/NcoI and ligated with the GUS geneobtained from pBI221 (Clontech Laboratories, Inc., Palo Alto, Calif.).The NcoI site is fused to the GUS gene such that the ATG of the NcoIsite functions as the start codon for the translation of the GUS gene.The CaMV 35S polyadenylation and termination signals are used for the 3'end of the chimeric gene.

Construction of pCIB3069 (35S-Adh1-GUS-35S)

pCIB246 is modified by adding the maize alcohol dehydrogenase gene Adh1intron number 1 (Adh1) (Dennis et al., Nucleic Acids Research,12:3983-4000 (1984)) into the Sal I site of pCIB246 to produce plasmidpCIB3007. The Adh1 intron is excised from the maize Adh1 gene as a BalI/Pst I fragment and subcloned into pUC 18 that was cut with Sma I/Pst Ito make a plasmid called Adh 1026. Adh 1026 is cut with Pvu II/Sac II,the fragments are made blunt ended with T4 DNA polymerase, Sal I linkersare added using standard procedures and a fragment of about 560 bp isrecovered from a 3% NuSeive gel and ligated into Sal I cut/phosphatasetreated pUC18. The Sal I linkered Adh intron #1 in the resulting plasmidis cut out with Sal I, gel purified, and ligated into Sal Icut/phosphatase treated pCIB246 to make plasmid pCIB3007.

pCIB3007 is cut with PstI and the ends made blunt by using T4 DNApolymerase (NEW England Biolabs) according to the suppliers'specifications. The resulting blunt ended molecules are cut with Sph Iand the approximately 5.8 Kb fragment with one blunt end and one Sph Iend is purified on a low gelling temperature (LGT) agarose gel usingstandard procedures. pCIB900 is cut with Sma I/Sph I and the fragmentcontaining the 35S/Bt gene is purified on a LGT agarose gel. The two gelpurified fragments are ligated in LGT agarose using T4 DNA ligaseaccording to standard conditions. The resulting ligated fragments aretransformed into E. coli using standard procedures and the resultingplasmid is called pCIB3062. There are two versions of pCIB3062.pCIB3062#1 has a Sma I site regenerated where the Sma I site and the T4polymerase blunted ends are ligated. This most likely results from theT4 polymerase nibbling a few base pairs from the Pst I site during theblunting reaction. pCIB3062#3 does not have this SmaI site.

pCIB3062#3 is cut with KpnI and made blunt-ended using T4 DNApolymerase, and subsequently cut with Pvu II to yield a 6.4 Kb fragmentwith blunt ends containing the 35S/GUS and 35S/Bt genes. This blunt-endfragment is ligated into Sma I cut pCIB3073 to produce pCIB3063 orpCIB3069. pCIB3069 contains the same fragment used to make pCIB3063, butthe chimeric genes in pCIB3069 are all in the same relative orientation,unlike those in pCIB3063. These plasmids contain a) a 35S promoteroperably linked to the hygromycin resistance gene; b) a 35S promoter,with Adh intron #1, operably linked to the GUS gene; and c) a 35Spromoter operably linked to a gene coding for the production of thesynthetic cryIA(b) insecticidal protein from Bacillus thuringiensis, asdescribed above. GUS Assays:

GUS assays are done essentially as described in Jefferson, Plant Mol.Bio. Reporter, 5:387-405 (1987). As shown above, plasmid pCIB246contains a CaMV 35S promoter fused with the GUS gene. The 5'untranslated leader of this chimeric gene contains a copy of the maizeAdh 1 intron #1. It is used here as a transformation control. Althoughthe same amount of pCIB246 is added to each transformation, thecalculated activity varied among Bt constructs tested. The valuesreported below are averages of 3 replicates. pCIB4407 was tested twice.

    ______________________________________                                        pCIB3069    28 nM MU/ug/min                                                   pCIB4407    0.7 nM MU/ug/min, 2.3 nM MU/ug/min                                ______________________________________                                    

Example 5A

ASSAY OF SYNTHETIC cryIA(b) gene FOR INSECTICIDAL ACTIVITY AGAINSTEUROPEAN CORN BORER

The synthetic cryIA(b) gene in pCIB4414 in E. coli is assayed forinsecticidal activity against European corn borer according to thefollowing protocol.

Molten artifical insect diet is poured into a 60 mm Gellman snap-cappetri dish. After solidification, E. coli cells, suspended in 0.1%Triton X-100, are spread over the surface at a concentration of 3×107cells/cm2. The plates are air dried. Ten first instar European cornborer, Ostrinia nubilalis, which are less than 12 hours old are thenplaced onto the diet surface. The test is incubated at 30° C. incomplete darkness for 2-5 days. At the end of the test percent mortalityis recorded. A positive clone has been defined as one giving 50% orhigher mortality when control E. coli cells give 0-10% backgroundmortality.

For comparison, the native cryIA(b) gene in pCIB3069 is tested at thesame concentration. Clones are tested at 3×107 cells/cm² diet; 20insects per clone.

The following results are observed:

    ______________________________________                                        Clone        Percent Mortality                                                ______________________________________                                        Control      0                                                                pCIB3069     100                                                              pCIB4414     100                                                              ______________________________________                                    

Thses results indicate that the insecticidal crystal protein produced bythe synthetic cryIA(b) gene demonstrates activity against European cornborer comparable to that of the IP produced by the native cryIA(b).Other plasmids containing a synthetic cryIA(b) gene were assayed in asimilar manner.

Example 5AB

ASSAY OF CRYIA(b) PROTEIN FOR INSECTICIDAL ACTIVITY AGAINST SUGARCANEBORER.

CryIA(b) was expressed in E. coli and assayed for insecticidal activityagainst Sugarcane borer (Diatrea saccharalis) according to the sameprotocol used for European corn borer, described immediately above. Theresults are summarized in the Table.

                  TABLE                                                           ______________________________________                                        SUGARCANE BORER ASSAY WITH Bt PROTEIN FROM E. COLI                            Protein        Percent                                                        Concentration  Mortality                                                      (ng/g)         CryIA(b)                                                       ______________________________________                                        10             0                                                              25             0                                                              50             7                                                              100            13                                                             250            40                                                             500            53                                                             1000           80                                                             LC50           380                                                            95% Cl         249-646                                                        ______________________________________                                    

The results indicate that the insecticidal protein produced by a maizeoptimized Bt gene is effective against Sugarcane borer. The upperconcentrations of CryIA(b) protein, 250 ng/g-1000 ng/g, are achievablein transgenic maize plants produced in accordance with the instantinvention.

Example 6

MAIZE PROTOPLAST ISOLATION AND TRANSFORMATION WITH THE SYNTHETIC BT GENE

Expression of the synthetic Bt gene is assayed in transientlytransformed maize protoplasts.

Protoplast Isolation Procedure:

1. The contents of 10 two day old maize 2717 Line 6 suspension culturesare pipetted into 50 ml sterile tubes and allowed to settle. All culturemedia is then removed and discarded.

2. Cells (3-5 ml Packed Cell Volume) are resuspended in 30 ml protoplastenzyme solution. Recipe follows:

3% Cellulase RS

1% Macerozyme R10 in KMC Buffer

KMC Buffer (recipe for 1 liter)

    ______________________________________                                               KCl       8.65 g                                                              MgCl.sub.2 --6H.sub.2 O                                                                16.47 g                                                              CaCl.sub.2 --2H.sub.2 O                                                                12.50 g                                                              MES       5.0 g                                                        ______________________________________                                         pH 5.6, filter sterilize                                                 

3. Mix cells well and aliquot into 100×25 mm petri dishes, about 15 mlper plate. Shake on a gyratory shaker for 4 hours to digest.

4. Pipette 10 ml KMC through each 100 micron sieve to be used. Filtercontents of dishes through sieve. Wash sieve with an equal volume KMC.

5. Pipette sieved protoplasts carefully into 50 ml tubes and spin in aBeckman TJ-6 centrifuge for 10 minutes at 1000 rpm (500×g).

6. Remove supernatant and resuspend pellet carefully in 10 ml KMC.Combine contents of 3 tubes into one and bring volume to 50 ml with KMC.

7. Spin and wash again by repeating the above step.

8. Resuspend all washed protoplasts in 50 ml KMC. Count in ahemocytometer. Spin protoplasts and resuspend at 8×10⁶ /ml inresuspending buffer (RS Buffer).

RS Buffer (recipe for 500 ml)

mannitol 27.33 g

CaCl2 (0.1 M stock) 75 ml

MES 0.5 g

pH 5.8, filter sterilize

Protoplast Transformation Procedure:

1. Aliquot 50 μg plasmid DNA (Bt IP constructs, both synthetic(pCIB4407) and native (pCIB3069)) to 15 ml polystyrene culture tubes.Also aliquot 25 μg GUS-containing plasmid DNA (which does not contain BtIP (pCIB246) to all tubes. 3 replications are used per construct to betested, with 1 rep containing no DNA as a control.

    ______________________________________                                        Bt constructs:      GUS construct:                                            ______________________________________                                        pCIB3069            pCIB246                                                   pCIB4407                                                                      ______________________________________                                    

2. Gently mix protoplasts well and aliquot 0.5 ml per tube.

3. Add 0.5 ml PEG-40 to each tube.

PEG-40:

0.4 M mannitol

0.1 M Ca(NO₃)₂ -4H₂ O

pH 8.0, filter sterilize

4. Mix gently to combine protoplasts with PEG. Wait 30 minutes.

5. Sequentially add 1 ml, 2 ml, and 5 ml W5 solution at 5 minuteintervals.

W5 Solution:

154 mM NaCl

125 mM CaCl₂ -H₂ O

5 mM KCl

5 mM glucose

pH 7.0, filter sterilize

6. Spin for 10 minutes in a Beckman TJ-6 centrifuge at about 1000 rpm(500 g).

Remove supernatant.

7. Gently resuspend pellet in 1.5 ml FW media and plate carefully in35×10 mm petri dishes.

FW media (recipe for 1 liter):

    ______________________________________                                               MS salts        4.3 g                                                         200X B5 vits.   5 ml                                                          sucrose         30 g                                                          proline         1.5 g                                                         mannitol        54 g                                                          2,4 D           3 mg                                                   ______________________________________                                         pH 5.7, filter sterilize                                                 

8. Incubate overnight in the dark at room temperature.

9. Perform GUS assays, insect bioassays, and ELISA's on protoplastextracts as described below.

Example 7

CONSTRUCTION OF A FULL-LENGTH SYNTHETIC MAIZE OPTIMIZED CRYIA(b) GENE

(SEQ ID NO: 4) shows the synthetic maize optimized sequence encoding thefull-length cryIA(b) insecticidal protein from B. thuringiensis. Thetruncated version described above represents the first approximately 2Kb of this gene. The remainder of the full-length gene is cloned usingthe procedures described above. Briefly, this procedure entailssynthesizing DNA oligomers of 40 to 90 NT in length, typically using 80mers as an average size. The oligomers are purified using standardprocedures of HPLC or recovery from a polyacrylamide gel. Purifiedoligomers are kinased and hybridized to form fragments of about 500 bp.The hybridized oligomers can be amplified using PCR under standardconditions. The 500 bp fragments, either directly from hybridizations,from PCR amplification, or recovered from agarose gels after eitherhybridization or PCR amplification, are then cloned into a plasmid andtransformed into E. coli using standard procedures. Recombinant plasmidscontaining the desired inserts are identified, as described above, usingPCR and/or standard miniscreen procedures. Inserts that appear correctbased upon their PCR and/or restriction enzyme profile are thensequenced to identify those clones containing the desired open readingframe. The fragments are then ligated together with the approximately 2Kb synthetic sequence described in Example 2 to produce a full-lengthmaize optimized synthetic cryIA(b) gene useful for expression of highlevels of CryIA(b) protein in maize.

G+C Content of native and synthetic Bt genes:

    ______________________________________                                        Full-length native                                                                             38.8%                                                        Truncated native 37.2%                                                        Full-length synthetic                                                                          64.8%                                                        Truncated synthetic                                                                            64.6%                                                        ______________________________________                                    

% homology of the final truncated version of the Bt gene relative to a"pure" maize codon usage gene: 98.25%

Example 8

Construction of a Plant Expressible, Full-length, Hybrid Partially MaizeOptimized CryIA(b) Gene.

pCIB4434 contains a full length CryIA(b) gene (SEQ ID NO: 8) comprisedof about 2 Kb of the synthetic maize optimized cryIA(b) gene with theremainder (COOH terminal encoding portion) of the gene derived from thenative gene. Thus, the coding region is a chimera between the syntheticgene and the native gene, but the resulting protein is identical to thenative cryIA(b) protein. The synthetic region is from nucleotide 1-1938(amino acids 1 to 646) and the native coding sequence is from nucleotide1939-3468 (amino acids 647 to 1155). The sequence of this gene is setforth in FIG. 7. A map of pCIB4434 is shown in FIG. 8.

The following oligos were designed to make pCIB4434:

KE134A28=5'-CGTGACCGAC TACCACATCG ATCAAGTATC CAATTTAGTT GAGT-3' (SEQ IDNO: 60)

KE135A28=5'-ACTCAACTAA ATTGGATACT TGATCGATGT GGTAGTCGGTC ACG-3' (SEQ IDNO: 61)

KE136A28=5'-GCAGATCTGA GCTCTTAGGT ACCCAATAGC GTAACGT-3' (SEQ ID NO: 62)

KE137A28=5'-GCTGATTATG CATCAGCCTAT-3' (SEQ ID NO: 63)

KE138A28=5'-GCAGATCTGA GCTCTTATTC CTCCATAAGA AGTAATTC-3' (SEQ ID NO: 64)

MK05A28=5'-CAAAGGTACC CAATAGCGTA ACG-3' (SEQ ID NO: 65)

MK35A28=5'-AACGAGGTGT ACATCGACCG-3' (SEQ ID NO: 66)

pCIB4434 is made using a four-way ligation with a 5.7 kb fragment frompCIB4418, a 346 bp Bst E II\Kpn I PCR-generated synthetic:native fusionfragment, a 108 bp Kpn I\Nsi I native CryIA(b) fragment from pCIB 1315,and a 224 bp Nsi I\Bgl II PCR-generated fragment. Standard conditionsfor ligation and transformation are as described previously.

A synthetic:native gene fusion fragment is made in two steps using PCR.The first 253 bp of the PCR fusion fragment is made using 100 pmols ofoligos KE134A28 and MK04A28 with approximately 200 ng of native cryIA(b)template in a 100 ul volume with 200 nm of each dNTP, 1×PCR buffer(Perkin Elmer Cetus), 20% glycerol, and 5 units of Taq polymerase(Perkin Elmer Cetus). The PCR reaction is run with the followingparameters: 1 minute at 94° C., 1 minute at 55° C., 45 seconds at 72°C., with extension 3 for 3 seconds for 25 cycles. A fraction (1%) ofthis first PCR reaction is used as a template along with 200 ng of thesynthetic cryIA(b) DNA to make the complete 351 bp synthetic:nativefusion fragment. Oligos used as PCR primers in this second PCR reactionare 50 pmols of MK35A28, 50 pmols of MK04A28, and 25 pmols of KE135A28.The PCR reaction mix and parameters are the same as those listed above.The resultant 351 bp synthetic:native fusion fragment is treated withProteinase K at 50 ugm\nl total concentration and phenol\chloroformextraction followed by ethanol precipitation before cutting with Bst EII\Kpn I using standard conditions.

The 224 bp Nsi I\Bgl II PCR fragment used in making pCIB4434 is madeusing 100 pmols of oligos KE137A28 and KE138A28 and 200 ng of the nativecryIA(b) gene as template in 100 ul volume with the same PCR reactionmix and parameters as listed above. The 230 bp PCR native cryIA(b)fragment is treated with Proteinase K, phenol\chloroform extracted, andethanol precipitated as described above, before cutting with Nsi I\BglII.

pCIB4434 was transformed into maize protoplasts as described above. Line6 2717 protoplasts were used with pCIB4434 and pCIB4419 as a control forcomparison. The results are shown below:

    ______________________________________                                                      ng Bt/mg protein                                                ______________________________________                                        4419(35S)     14,400 ± 2,100                                               434(full-length)                                                                            2,200 ± 900                                                  ______________________________________                                         Background = 13 ng Bt/mg protein for untransformed protoplasts           

The results indicate that pCIB4434 expresses at a level of about 15% ofpCIB4419.

Western blot analysis shows at least one-third of the cryLA(b) proteinproduced by pCIB4434 in this system is about 130 kD in size. Therefore,a significant amount of full-length cryIA(b) protein is produced inmaize cells from the expression of pCIB4434.

Example 7

Construction of a Full-Length, CryIA(b) Genes Encoding aTemperature-Stable CryIA(b) Protein.

Constructs pCIB5511-5515, each containing a full-length, cryIA(b) geneare described below. In these sequences, the 26 amino acid deletionbetween amino acids 793 and 794, KCGEPNRCAPHLEWNPDLDCSCRDGE (see: SEQ IDNOS: 8, 10, 12, 14, 16), present in cryIA(a) and cryIA(c) but not incryIA(b), has been repaired. The gene in pCIB5513 is synthetic; theother four genes are hybrids, and thus are partially maize optimized.

Construction of pCIB5511

This plasmid is a derivative of pCIB4434. A map of pCIB5511 is shown inFIG. 10. A 435 bp segment of DNA between bp 2165 and 2590 wasconstructed by hybridization of synthetic oligomers designed torepresent the upper and lower strand as described above for theconstruction of the truncated cryIA(b) gene. This segment of syntheticDNA is synthesized using standard techniques known in the art andincludes the 26 amino acid deletion found to occur naturally in thecryIA(b) protein in Bacillus thuringiensis kurstaki HD-1. The entireinserted segment of DNA uses maize optimized codon preferences to encodeamino acids. The 26 amino acids used to repair the naturally occurringdeletion are contained within this fragment. They are inserted startingat position 2387 between the KpnI site at nt 2170 and the XbaI site atnt 2508 (2586 in pCIB551 1) of pCIB4434. pCIB5511 is constructed via athree way ligation using a 3.2 Kb fragment obtained by restrictiondigestion of pCIB4434 with SphI and KpnI, a 3.8 Kb fragment obtained bydigestion of pCIB4434 with SphI and XbaI, and a 416 bp fragment obtainedby digestion of the synthetic DNA described above, with KpnI and XbaI.Enzymatic reactions are carried out under standard conditions. Afterligation, the DNA mixture is transformed into competent E. coli cellsusing standard procedures. Transformants are selected on L-agarcontaining 100 μg/ml ampicillin. Plasmids in transformants arecharacterized using standard mini-screen procedures. The sequence of therepaired cryIA(b) gene encoding the cryIA(b) temperature (heat) stableprotein is set forth in FIG. 9 (SEQ ID NO: 10).

Construction of pCIB5512

This plasmid construct is a derivative of pCIB4434. A map of pCIB5512 isshown in FIG. 12. DNA to repair the 26 amino acid deletion is preparedusing standard techniques of DNA synthesis and enzymatic reaction. Threedouble stranded DNA cassettes, pGFcas1, pGFcas2 and pGFcas3, each about300 bp in size, are prepared. These cassettes are designed to containthe maize optimized codons while maintaining 100% amino acid identitywith the insecticidal protein. These cassettes are used to replace theregion between restriction site BstEII at position 1824 and XbaI atposition 2508 and include the insertion of the additional 78 bp whichencode the missing 26 amino acids (described above for pCIB5511 inpCIB4434). Each of these cassettes is cloned into the EcoRV site of thevector Bluescript (Stratagene) by standard techniques. The threecassettes are designed to contain overlapping restriction sites.Cassette 1 has restriction sites BstEII at the 5' end and EcoRV at the3' end: cassette 2 has EcoRV at the 5' end and ClaI at the 3' end andcassette 3 has ClaI at the 5' end and Xba I at the 3' end. They arecloned individually in Bluescript and the the complete 762 bp fragmentis subsequently assembled by ligation using standard techniques.pCIB5512 is assembled using this 762 bp fragment and ligating it with a6.65 Kb fragment obtained by a complete digestion of pCIB4434 with BstEIand a partial digestion with XbaI. Alternatively, a four way ligationusing the same vector and the three cassettes digested with the specificenzymes can be employed. Enzymatic reactions are carried out understandard conditions. After ligation, the DNA mixture is transformed intocompetent E. coli cells using standard procedures. Transformants areselected on L-agar containing 100 μg/ml ampicillin. Plasmids intransformants are characterized using standard mini-screen procedures.The resulting plasmid is pCIB5512. The sequence of the repaired cryIA(b)gene is illustrated in FIG. 11 (SEQ ID NO: 12). This repaired cryIA(b)differs from that carried in pCIB5511 in that a larger region of thecryIA(b) coding region is optimized for maize expression by using maizepreferred codons.

Construction of pCIB5513

This plasmid contains a repaired cryIA(b) gene derived from pCIB5512. Amap of pCIB5513 is shown in FIG. 14. The region 3' from the XbaI site atposition 2586 to the end of the gene (BglII site at position 3572) isreplaced entirely with maize optimized codons. This region issynthesized, using standard techniques of DNA synthesis and enzymaticreaction, well known in the art, as four double stranded DNA cassettes(cassettes # 4, 5, 6, 7). Adjacent cassettes have overlappingrestriction sites to facilitate assembly between cassettes. These areXbaI and XhoI at the 5' and 3' ends of cassette 4; XhoI and SacI at the5' and 3' ends, respectively, of cassette 5; SacI and BstXI at the 5'and 3' ends, respectively, of cassette 6; and BstXI and BglII at the 5'and 3' ends, respectively, of cassette 7. As described for pCIB5512, thecassettes are cloned into the blunt-end EcoRV site of the Bluescriptvector (Stratagene) and the full-length "repaired" cryIA(b) gene clonedeither by sequential assembly of the above cassettes in Bluescriptfollowed by ligation of the complete 967 bp synthetic region with a 6448bp fragment obtained by a complete digestion of pCIB5512 with BglII anda partial digestion with XbaI. Alternately, the plasmid containing thefull-length genes is obtained by a 5-way ligation of each of the fourcassettes (after cleavage with the appropriate enzymes) and the samevector as above. The sequence of the full-length, "repaired" cryIA(b)gene is set forth in FIG. 13 (SEQ ID NO: 14). The protein encoded by thevarious synthetic and synthetic/native coding region chimeras encode thesame protein. This protein is the heat-stable version of cryIA(b)produced by repairing the naturally occurring 26 amino acid deletionfound in the cryIA(b) gene from Bacillus thuringiensis kurstaki HD-1when the homologous region is compared with either cryIA(a) or cryIA(c)Bacillus thuringiensis delta-endotoxins.

Construction of pCIB5514

This plasmid is a derivative of pCIB4434. A map of pCIB5514 is shown inFIG. 16. It is made using synthetic DNA cassette #3 (see above) whichcontains a maize optimized sequence of the region between the ClaI site(position 2396) found in the 26 amino acid thermostable region and theXbaI site at position 2508 in pCIB4434 (2586 in pCIB5511). The regionbetween nt 2113 of pCIB4434 and the junction of the thermostable regionis PCR amplified by using pCIB4434 as template with the followingprimers:

forward: 5'GCACCGATATCACCATCCAAGGAGGCGATGACGTATTCAAAG-3' (SEQ ID NO: 67)

reverse:

5'-AGCGCATCGATTCGGCTCCCCGCACTTGCCGATTGGACTTGGGGCTGAAAG-3' (SEQ ID NO:68).

The PCR product is then digested with restriction enzymes KpnI and ClaIand ligated in a four part reaction with a 189 bp fragment obtained bydigestion of cassette 3 with ClaI and XbaI, a 3.2 Kb fragment ofpCIB4434 digested with SphI and KpnI, and a 3.8 Kb fragment of pCIB4434obtained by digestion with SphI and Xba. Enzymatic reactions are carriedout under standard conditions. The ligation product is transformed intocompetent E coli cells, selected with ampicillin and screened usingstandard procedures described above. The sequence of the repairedcryIA(b) gene contained in pCIB5514 is shown in FIG. 15 (SEQ ID NO: 16).

pCIB4434 was modified by adding the 78bp Geiser thermostable element(Geiser TSE), described above, between the Kpn I site (2170 bp) and theXba I site (2508 bp) in the native Btk region. The exact insertion sitestarts at the nucleotide #2379. The region containing the Geiser TSE wasamplified by two sets of PCR reactions, i.e. the Kpn I-Geiser TSEfragment and the Geiser TSE-Xba I fragment.

PCR primer# 1: (Kpn I site)

5'-ATTACGTTAC GCTATTGGGT ACCTTTGATG-3' (SEQ ID NO: 69)

PCR primer#2: (Geiser TSE bottom)

                          (SEQ ID NO:70)                                          5'-TCCCCGTCCC TGCAGCTGCA GTCTAGGTCC GGGTTCCACT                                      CCAGGTGCGG AGCGCATCGA TTCGGCTCCC CGCACTTGCC                                   GATTGGACTT GGGGCTGA-3'                                              

PCR primer#3: (Geiser TSE top)

                          (SEQ ID NO:71)                                          5'CAAGTGCGGG GAGCCGAATC GATGCGCTCC GCACCTGGAG                                    TGGAACCCGG ACCTAGACTG CAGCTGCAGG GACGGGGAAA                                   AATGTGCCCA TCATTCCC-3'                                                 

PCR primer#4: (Xba I site)

5'-TGGTTTCTCT TCGAGAAATT CTAGATTTCC-3' (SEQ ID NO: 72)

After the amplification, the PCR fragments were digested with (Kpn I+ClaI) and (Cla I+Xba I), respectively. These two fragments were ligated tothe Kpn I and Xba I digested pCIB4434. The resulting construct pCIB5515is pCIB4434 with a Geiser TSE and an extra Cla I site flanked by Kpn Iand Xba I. A map of pCIB5515 is illustrated in FIG. 38. The cryIA(b)gene contained herein, which encodes a temperature stable cryIA(b)protein, is shown in FIG. 37 (SEQ ID NO: 27).

Examples 9-20 set forth below are directed to the isolation andcharacterization of a pith-preferred promoter.

Example 9

RNA Isolation and Northern Blots

All RNA was isolated from plants grown under greenhouse conditions.Total RNA was isolated as described in Kramer et al., Plant Physiol.,90:1214-1220 (1990) from the following tissues of Funk maize line 5N984:8, 11, 15, 25, 35, 40, and 60 day old green leaves; 8, 11, 15, 25, 35,39, 46, 60 and 70 day old pith; 60 and 70 day old brace roots from Funkmaize line 5N984; 60 and 70 day 5N984 sheath and ear stock. RNA was alsoisolated from 14 day 211D roots and from developing seed at weeklyintervals for weeks one through five post-pollenation. Poly A+RNA wasisolated using oligo-dT as described by Sambrook et al., MolecularCloning: A Laboratory Manual (2nd ed.), 1989, and Northern blots werecarried out, also as per Sambrook et al. using either total RNA (30 μg)or poly A+RNA (2-10 μg). After electrophoresis, RNA was blotted ontoNitroplus 2000 membranes (Micron Separations Inc). The RNA was linked tothe filter using the Stratalinker (Stratagene) at 0.2 mjoules. Thenortherns were probed with the 1200 bp EcoRI pith (TRpA) 8-2 cDNAfragment, isolated by using 0.8% low melting temperature agarose in aTBE buffer system. Northerns were hybridized and washed and the filtersexposed to film as described in Isolation of cDNA clones.

Example 10

Isolation of cDNA Clones

First strand cDNA synthesis was carried out using the BRL AMV reversetranscriptase system I using conditions specified by the supplier (LifeTechnologies, Inc., Gaithersburg, Md.). Specifically, 25 μl reactionscontaining 50 mM Tris-HCl pH 8.3, 20 mM KCl, 1 mM DTT, 6 mM MgCl2, 1 mMeach of each dNTP, 0.1 mM oligo (dT)12-18, 2 μg pith poly(A+) RNA, 100μg/ml BSA, 50 μg/ml actinomycin D, 8 units placental RNase inhibitor, 1μl (10 mM Ci/ml) 32P dCTP>3000 mCi/mM as tracer, and 30 units AMVreverse transcriptase were incubated at 42° C. for 30 min. AdditionalKCl was added to a concentration of 50 mM and incubation continued afurther 30 min. at 42° C. KCl was added again to yield a finalconcentration of 100 mM. Additional AMV reverse transcriptase reactionbuffer was added to maintain starting concentrations of the othercomponents plus an additional 10 units, and the incubation continued at42° C. for another 30 min. Second strand synthesis was completed usingthe Riboclone cDNA synthesis system with Eco RI linkers (Promega,Madison, Wis.). Double stranded cDNA was sized on an 1% agarose gelusing Tris-borate-EDTA buffer as disclosed in Sambrook et al., andshowed an average size of about 1.2 Kb. The cDNA was size fractionatedusing NA45 DEAE membrane so as to retain those molecules of about 1000bp or larger using conditions specified by the supplier (Schleicher andSchuell). Size fractionated cDNA was ligated into the Lambda ZapIIvector (Stratagene, La Jolla, Calif.) and packaged into lambda particlesusing Gigapack II Plus (Stratagene, La Jolla, Calif.). The unamplifiedlibrary had a titer of 315,000 pfu while the amplified library had atiter of 3.5 billion/ml using PLK-F cells.

Recombinant phage were plated at a density of 5000 pfu on 150×15 mmL-agar plates. A total of 50,000 phage were screened using duplicatelifts from each plate and probes of first strand cDNA generated fromeither pith derived mRNA or seed derived mRNA. The lifts were done asdescribed in Sambrook et al. using nitrocellulose filters. DNA was fixedto the filters by UV crosslinking using a Stratalinker (Stratagene, LaJolla, Calif.) at 0.2 mJoule. Prehybridization and hybridization of thefilter were carried out in a solution of 10×Denhardts solution, 150μg/ml sheared salmon sperm DNA, 1% SDS, 50 mM sodium phosphate pH 7, 5mM EDTA, 6×SSC, 0.05% sodium pyrophosphate. Prehybridization was at 62°C. for 4 hours and hybridization was at 62° C. for 18 hours (overnight)with 1 million cpm/ml in a volume of 40 ml. Filters were washed in 500ml of 2×SSC, 0.5% SDS at room temperature for 15 min. then at 63° C. in0.1×SSC, 0.5% SDS for 30 min. for each wash. Radiolabeled DNA probeswere made using a BRL random prime labeling system and unincorporatedcounts removed using Nick Columns (Pharmacia). Filters were exposedovernight to Kodak X-Omat AR X-ray film with (DuPont) Cronex LightningPlus intensifying screens at -80° C. Plaques showing hybridization withthe pith-derived probe and not the seed-derived probe were plaquepurified for further characterization.

Example 11

Isolation of Genomic Clones

Genomic DNA from Funk inbred maize line 211D was isolated as describedby Shure et al., Cell, 35:225-233 (1988). The DNA was partially digestedwith Sau 3A and subsequently size fractionated on 10-40% sucrosegradients centrifuged in a Beckman SW40 rotor at 22,000 rpm for 20 hoursat 20° C. Fractions in the range of 9-23 Kb were pooled and ethanolprecipitated. Lambda Dash II (Stratagene) cut with Bam HI was used asdescribed by the supplier. The library was screened unamplified and atotal of 300,000 pfu were screened using the conditions described above.The library was probed using pith-specific (TrpA) cDNA clone 8-2,pCIB5600 which was identified in the differential screen of the cDNAlibrary. Isolated clones were plaque purified and a large scale phagepreparation was made using Lambdasorb (Promega) as described by thesupplier. Isolated genomic clones were digested with Eco RI and the 4.8kb EcoRI fragment was subcloned into Bluescript vector (Stratagene).

Example 12

DNA Sequence and Computer Analysis

Nucleotide sequencing was performed using the dideoxy chain-terminationmethod disclosed in Sanger et al., PNAS,74:5463-5467 (1977). Sequencingprimers were synthesized on an Applied Biosystems model 380B DNAsynthesizer using standard conditions. Sequencing reactions were carriedout using the Sequenase system (US Biochemical Corp.). Gel analysis wasperformed on 40 cm gels of 6% polyacrylamide with 7 M urea inTris-Borate-EDTA buffer (BRL Gel-Mix 6). Analysis of sequences andcomparison with sequences in GenBank were done using the U. of WisconsinGenetic Computer Group Sequence Analysis Software (UWGCG).

Example 13

Mapping the Transcriptional Start Site

Primer extension was carried according to the procedure of Metraux etal., PNAS,86:896-900 (1988). Briefly, 30 μg of maize pith total RNA wereannealed with the primer in 50 mM Tris pH 7.5,40 mM KCl, 3 mM MgCl2 (RTbuffer) by heating to 80° C. for 10 minutes and slow cooling to 42° C.The RNA/primer mix was allowed to hybridize overnight. Additional RTbuffer, DTT to 6 mM, BSA to 0.1 mg/ml, RNAsin at 4 U/ml and dNTP's at 1mM each were added. Then 8 units AMV reverse transcriptase were addedand reaction placed at 37° C. for one hour. The primer used was5'-CCGTTCGTTC CTCCTTCGTC GAGG-3' (SEQ ID NO: 73), which starts at +90 bprelative to the transcription start. See FIG. 29A. A sequencing ladderusing the same primer as in the primer extension reaction was generatedusing the 4.8 Kb genomic clone to allow determination of thetranscriptional start site. The sequencing reaction was carried out asdescribed in Example 12.

RNase protection was used to determine if the the 371 bp sequence from+2 bp to +373 bp (start of cDNA) was contiguous or if it contained oneor more introns. A 385 bp SphI-NcoI fragment spanning +2 bp to +387 bprelative to transcriptional start see FIG. 29B was cloned intopGEM-5Zf(+) (Promega) and transcribed using the Riboprobe Gemini system(Promega) from the SP6 promoter to generate radioactive antisense RNAprobes as described by the supplier. RNase protection was carried out asdescribed in Sambrook et al. pBR322 (cut with HpaII and end labelledwith 32P-dCTP) and Klenow fragment were used molecular weight markers.Gels were 6% acrylamidene/7M urea (BRL Gel-Mix 6) and were run at 60watts constant power.

Example 14

Genomic Southern Blots

Genomic DNA was isolated from maize line 211D using the procedure ofShure et al., supra. 8 μg of genomic DNA were used for each restrictionenzyme digest. The following enzymes were used in the buffer suggestedby the supplier: BamHI, EcoRI, EcoRV, HindIII, and SacI. Pith cDNA clonenumber 8-2 was used for estimating gene copy number. The digested DNAwas run on a 0.7% agarose gel using Tris-Borate-EDTA buffer system. Thegel was pretreated with 250 mM HCl for 15 min. to facilitate transfer ofhigh molecular weight DNA. The DNA was transferred to Nitroplus 2000membrane and subsequently probed with the pith cDNA 8-2. The blot waswashed as described in Example 10.

Example 15

PCR Material and Methods

PCR reactions were preformed using the GeneAmp DNA Amplication reagentkit and AmpliTaq recombinant Taq DNA polmerase (Perkin Elmer Cetus).Reaction condition were as follows: 0.1 to 0.5 uM of each of the twoprimers used per reaction, 25 ng of the pith 4.8 Kb EcoRI fragment inBluescript, plus the PCR reaction mix described by the supplier for atotal volume of 50 uL in 0.5 mL GeneAmp reaction tube (Perkin ElmerCetus). The DNA Thermal Cycler (Perkin Elmer Cetus) using the Step-Cycleprogram set to denature at 94° C. for 60 s, anneal at 55° C. for 60 s,and extend at 72° C. for 45 s followed by a 3-s-per-cycle extension fora total of 30 cycles. The following primer sets were used: I. 83×84,-429 bp to -2 bp; II. 49×73, -69 bp to +91 bp; III. 38×41, +136 bp to+258 bp; and IV. 40×75, +239 bp to +375 bp. These are marked on FIG. 24.

Example 16

Isolation of a Pith-Preferred Gene.

A cDNA library derived from pith mRNA cloned into Lambda Zap andscreened using first strand cDNA derived from either pith or seed mRNA.Clones which hybridized with only the pith probe were plaque purifiedand again screened. Clones passing the second screen were used as probesin northern blots containing RNA from various maize tissues.

Example 17

Gene Structure and Sequence Analysis.

The 1.2 Kb insert of the cDNA clone 8-2 was sequenced using the dideoxymethod of Sanger et al., supra. Likewise, the genomic equivalentcontained on a 4.8 Kb EcoRI fragment in Bluescript denoted as pCIB5601,was sequenced. This information revealed that the genomic copy of thecoding region spans 1.7 Kb and contains five introns. The mRNAtranscript represents six exons. This is shown in FIG. 24. The exonsrange in size from 43 bp to 313 bp and the introns vary in size from 76bp to 130 bp. The entire sequence of the gene and its correspondingdeduced amino acid sequence are shown in FIG. 24 (SEQ ID NOS: 18 and19).

This gene encodes a protein of 346 amino acids with a molecular mass ofabout 38 kD. As illustrated in Table 1, the predicted protein shows 62%similarity and 41% identity with the subunit protein of Pseudomonasaeruginosa and has high homology with trpA proteins from otherorganisms.

                  TABLE 1                                                         ______________________________________                                        Conservation of TrpA sequences between a maize TrpA gene and other            organisms.                                                                    Organisms        % amino acid                                                                             % amino acid                                      compared         Similarity Identity                                          ______________________________________                                        Haloferax volancii                                                                             56.4       36.1                                              Methanococcus voltae                                                                           58.1       35.1                                              Pseudomonas aeruginosa                                                                         62.5       41.8                                              Neurospora crassa                                                                              61.4       39.3                                              Saccharomyces cerevisiae                                                                       56.7       36.1                                              ______________________________________                                         Similarity groupings, I = L = M = V, D = E, F = Y, K = R, N = Q, S = T        Similarities and identities were done using the GAP program from UWGCG.  

Crawford et al., Ann. Rev. Microbiol., 43:567-600 (1989), incorporatedherein by reference, found regions of conserved amino acids in bacterialtrpA genes. These are amino acids 49 to 58, amino acids 181 to 184, andamino acids 213 to 216, with the rest of the gene showing greatervariability than is seen in the TrpB sequence. An alignment of knowntrpA proteins with the maize TrpA protein (not shown) illustrates thatthe homology between the maize gene and other trpA proteins isconsiderable. Also, it is comparable to the level of homology observedwhen other TrpA proteins are compared to each other as described inCrawford et al., supra.

To determine the location of the transcription start site and whether ornot there were introns present in this region, four polymerase chainreaction (PCR) generated fragments of about 122 bp to 427 bp from theregion -429 bp to +372 bp were used for northern analysis. The resultsof the northerns showed that PCR probes II, III, IV hybridized to pithtotal RNA and PCR probe I did not hybridize. This indicated that thetranscription start was in the -69 bp to +90 bp region. To moreprecisely locate the transcriptional start site, primer extension wasemployed. FIG. 28A shows that when a primer (#73) located at +90 bprelative to the transcriptional start is used for primer extension, thetranscriptional start site is located at +1, 1726 bp on the genomicsequence.

The first ATG from the transcriptional start site is at +114 bp. This isthe ATG that would be expected to serve as the site for translationalinitiation. This ATG begins an open reading that runs into the openreading frame found in the cDNA clone. The first 60 amino acids of thispredicted open reading frame strongly resemble a chloroplast transitpeptide. See Berlyn et al. PNAS, 86:4604-4608 (1989) and Neumann-Karlinet al., EMBO J., 5:9-13 (1986). This result suggests that this proteinis targeted to a plastid and is likely processed to yield the activeprotein. Transient expression assays in a maize mesophyll protoplastsystem using a maize optimized B.t. gene driven by the trpA promotershowed that when the ATG at +114 bp is used as the fusion point, thehighest levels of expression are obtained. Using either of the next twoATGs in the sequence substantially reduces the level of expression ofthe reporter gene. The ATG at +390 bp gave some activity, but at a muchlower level than the +114 ATG, and the ATG at +201 bp gave no activity.

Athough a number of TATA like boxes are located upstream of the upstreamof the transcriptional start site at +1 bp, the TATAAT at -132 bp ismost like the plant consensus of TATAAA. See Joshi, Nuc. Acids Res.,15:6643-6653 (1987). The presumptive CCAAT like box was found at -231 bp. The nucleotide sequence surrounding the ATG start (GCGACATGGC; see SEQID NO: 18) has homology to other maize translation starts as describedin Messing et al., Genetic Engineering of Plants: An AgriculturalPerspective, Plenum Press, pp. 211-227 (1983), but differs from thatconsidered a consensus sequence in plants (ANNATGGC). See, Joshi, above.The presumptive poly(A) addition signal is located at 3719 bp (AATAAA)on the genomic sequence, 52 bp from the end of the cDNA. The sequencematches known sequences for maize as described in Dean et al., Nuc.Acids Res., 14:2229-2240 (1986), and is located 346 bp downstream fromthe end of protein translation. See Dean et al., Nuc. Acids Res.,14:2229-2240 (1986). The 3' untranslated sequence of the cDNA ends at3775 bp on the genomic sequence.

FIG. 27 shows a Southern blot of maize 211D genomic DNA with theapproximate gene copy number as reconstructed using pith gene 8-2 cDNA.From the restriction digests and reconstruction there appear to be 1-2copies of the gene present per haploid genome. There do not appear to beother genes with lower levels of homology with this gene. Therefore,this represents a unique or small member gene family in maize.

Example 18

RNase Protection

The structure of the 5' end of the mRNA was determined using RNaseprotection. The RNase protection was carried out using a proberepresenting 385 nt from +2 bp to +387 bp. This region from the genomicclone was placed in the RNA transcription vector pGEM-5Zf(+) and a 32Plabelled RNA probe generated using SP6 polymerase. The probe and theextra bases from the multiple cloning site produce a transcript of 461nt. The probe was hybridized with total pith RNA and subsequentlydigested with a mixture of RNase A and T1 and the protected fragmentsanalyzed on denaturing polyacrylamide gels. Analysis of the gels shows aprotected fragment of about 355 nt and another fragment of about 160 nt.See FIG. 28B.

The fact that primer extension using a primer (#73) at +80 bp produces aproduct of 90 NT in length argues that the 5' end of the transcript islocated at position +1 bp. Primer extension from a primer in this regionproduces a product, so one would expect this also to be detected by theRNase protection assay. This primer is located in the 5' region of theRNase protection probe. The cDNA clone contains sequences present in the3' end of the RNase protection probe and hence were expected to beprotected in this assay. Since only one band is present on the gel whichcould account for both of these sequences, we are confident that theprotected fragment is indeed the larger band and that the smaller singleband is an artifact. If there were an intron in this region, fragmentsfrom each end would be present in the probe, and hence would bedetectable on the gel. Of the two bands seen, one of them appears torepresent the entire 5' region, therefore we do not believe that thereis an intron located in this region.

Example 19

Complementation of E. coli TrpA Mutant with the Pith cDNA 8-2

E. coli strain CGSC strain 5531 from the E. coli Genetic Stock Center,Yale University (O. H. Smith lab strain designation, #M5004) withchromosomal markers glnA3, TrpA9825, 1-,IN(rrnD-rrnE), thi-1 asdescribed in Mayer et al., Mol. Gen. Gentet., 137:131-142 (1975), wastransformed with either the pith (TRpA) cDNA 8-2 or Bluescript plasmid(Stratagene) as described in Sambrook et al., supra. The transformantscontaining the TrpA cDNA 8-2 had the ability to grow without thepresence of tryptophan on minimal medium whereas the transformants withthe Bluescript (Stratagene) plasmid or untransformed control were notable to grow without tryptophan. The cells transformed with the maizeTrpA gene grew very slowly with colonies visible after seven days growthat room temperature. All strains were grown on M9 minimal mediumsupplemented with 200 ug/ml glutamine, 0.01 ug/ml thiamine and with orwithout 20 ug/ml tryptophan. All transformants were checked for thepresence of the appropriate plasmid by restriction enzyme analysis.Colonies growing in the absence of tryptophan all contained clone 8-2containing the cDNA for the putative maize TrpA gene, as confirmed bySouthern hybridization (data not shown). These results support theconclusion that this is the maize tryptophan synthase subunit A protein.

Example 20

Gene Expression

The expression pattern of the pith-preferential gene throughout theplant was examined. Different maize genotypes were also examined forpatterns of expression of this gene. The following tissues were used asthe source of RNA for these studies: upper, middle, and lower pith,brace roots, ear shank, cob in genotype 5N984; upper, middle, lowerpith, 10 day old leaves, 14 day old roots and pith from the entire plantin genotype 211D, and seed from genotype 211D which had been harvestedat weekly intervals one to five weeks post-pollination. Lower pith isderived from, i.e. constitutes the two internodes above brace roots;middle pith is derived from the next three internodes; upper pithrepresents the last two internodes before the tassel in 60 and 70 dayplants. Only two internodes were present in 39 day old plants and threeinternodes for 46 day old plants. Northern blot analysis shows thattranscripts hybridizing with a probe derived from the pith cDNAaccumulate rapidly in young pith and young leaf. As the age of the plantincreases and one moves up the stalk, there is a significant decrease inthe amount of transcript detected. See FIGS. 25A-D. At no time ismessage from this gene detected in seed derived RNA, either total RNA orpoly A+RNA. See FIG. 26. Transcript is also detected in root, earshank,and sheath but not at the high levels detected in the pith and youngleaf tissues. See FIGS. 25B, 25C. Some message is detected in braceroots, but only at a very low level. See FIG. 25D. Six maizeundifferentiated callus lines were analyzed by northern blot analysisand no expression was found for this gene (data not shown) in any callussample. The level of expression of this gene is extremely high since avery strong signal to a probe from TrpA gene 8-2 can be detected in pithand leaf as little as two hours after exposure of the blot to film (FIG.25A). The amount of mRNA made is comparable to that derived from themaize phosphoenolpyruvate carboxylase gene disclosed in Hudspeth et al.,Plant Mol. Biology, 12:579-589 (1989), another highly expressed maizegene. Hudspeth is incorporated herein by reference.

The expression pattern of this gene is not temporally constant.Expression is very high in the lower and middle pith of plants less than60 days old and decreases rapidly near the top of the plant. As theplant reaches maturity, e.g. over 70 days old, the expression drops tonearly undetectable levels except in the lower pith and earshank. Theaccumulation of transcript in young leaf is nearly as high as that seenin lower pith but expression decreases rapidly and is undetectable inleaves over 40 days of age. Expression in leaf was found to be variabledepending on the season when it is grown.

Examples 21-39 set forth below are directed to the isolation,characterization and expression analysis of a pollen-specific promoteraccording to the present invention.

Identification of Pollen-Specific Proteins Example 21

Maize Plant Growth

Maize plants (Zea mays Funk inbred 211D) were grown from seed in avermiculite/sand mixture in a greenhouse under a 16 hour light/8 hourdark regime.

Example 22

Total Pollen Protein Isolation

Mature pollen was isolated from maize plants at the time of maximumpollen shed. It was sieved to remove debris, frozen in liquid nitrogen,and a 3-4 ml volume of frozen pollen was ground in a mortar and pestlewith an equal volume of 75-150 μm glass beads. 40 ml of grinding buffer(2 mM EDTA, 5 mM DTT, 0.1% SDS, 100 mM Hepes pH 8) was added and themixture was ground again. The glass beads and intact pollen grains werepelleted by low speed centrifugation, and mixture was clarified bycentrifugation at 10,000 g for 15 minutes. Protein was precipitated fromthe supernatant by addition of acetone to 90%.

Example 23

Pollen Exine Protein Isolation

Exine Protein was isolated from maize 211D shed pollen as described inMatousek and Tupy, J., Plant Physiology 119:169-178 (1985).

Example 24

Leaf Protein Isolation

Young leaves (about 60% expanded) were cut from the maize plant themidrib removed. Total protein was isolated as for pollen, except thatthe material was not frozen and grinding was in a Waring blender withoutglass beads.

Example 25

Kernel Protein Isolation

Ears with fully developed, but still moist kernels were removed from theplant and the kernels cut off with a scalpel. Total protein was isolatedas for leaves.

Example 26

Gel Electrophoresis of Maize Proteins

Pollen, leaf and kernel proteins were separated on SDS polyacrylamidegels as described in Sambrook et al, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press: New York (1989). Followingstaining by Coomasie blue, protein bands from pollen, leaf and kernelwere compared and abundant proteins of approximately 10 kD, 13 kD, 20kD, 45 kD, 55 kD and 57 kD were determined to be pollen specific.

Identification of Pollen-Specific cDNA Clones Example 27

Partial Sequence Determination of Pollen-Specific Proteins

Protein bands determined to be pollen-specific were purified byelectroblotting from the polyacrylamide gel onto PVDF membrane(Matsudaira, P., J. Biol. Chem. 261:10035-10038 (1987)) or by reversephase HPLC. N-terminal sequence of the purified proteins was determinedby automated Edman egradation with an Applied Biosystems 470A gas-phasesequencer. Phenylthiohydantoin (PTH) amino acids were identified usingan Applied Biosystems 120A PTH analyzer. To obtain internal sequence,proteins were digested with endoproteinase Lys-C (Boehringer Mannheim)in 0.1 M Tris-HCl, pH 8.5, for 24 hours at room temperature using anenzyme:substrate ratio of 1:10. Resulting peptides were isolated by HPLCusing an Aquapore C-8 column eluted with a linearacetonitrile/isopropanol (1 :1 ratio) gradient (0 to 60%) in 0.1% TFA.Sequence of isolated Lys-C peptides was determined as above. Thefollowing sequences were determined for the 13 kD pollen-specificprotein:

N-terminus: TTPLTFQVGKGSKPGHLILTPNVATI (SEQ ID NO: 74)

LysC 61: KPGHLILTPNVATISDVVIK (SEQ ID NO: 75)

LysC 54: SGGTRIADDVIPADFK (SEQ ID NO: 76)

LysC 49: EHGGDDFSFFLK (SEQ ID NO: 77)

LysC 43: EGPTGTWTLDTK (SEQ ID NO: 78)

Example 28

Synthesis of Oligonucleotide Probes for Pollen-Specific cDNAs

Regions of peptide sequence in the 13 kD protein with low codonredundancy were selected, and suitable oligonucleotide probes for thegene encoding these regions were synthesized on an Applied Biosystems380A synthesizer. The following oligonucleotides were synthesized:

                          (SEQ ID NO:79)                                          Oligo #51 5'-AA RTC RTC ABC ACC RTG YTC-3'                                                           (SEQ ID NO:80)                                         Oligo #58 5'-CC YTT NCC CAC YTG RAA-3'                                    

where the columns of nucleotides represent bases that were incorporatedrandomly in equal proportions at the indicated position in the oligo.Oligo #51 encodes the amino acid sequence EHGGDDF (amino acids 1 to 7 ofSEQ ID NO: 77) found in peptide LysC 49, and Oligo #58 encodes the aminoacid sequence FQVGKG (amino acids 6 to 11 of SEQ ID NO: 74) found inpeptide N-terminus. Use of these mixed oligonucleotides to screen a cDNAlibrary for the pollen-specific gene will be described below.

Example 29

Construction of a Maize Pollen cDNA Library

Total maize RNA from maize 211D shed pollen was isolated as described inGlisen et al, Biochemistry 13:2633-2637 (1974). Poly A+mRNA was purifiedfrom total RNA as described in Sambrook et al. Using this mRNA, cDNA wasprepared using a cDNA synthesis kit purchased from Promega, followingprotocols supplied with the kit. The EcoRI linkers were added to thecDNA and it was ligated into arms of the cloning vector lambda Zap,purchased from Stratagene and using the protocol supplied by themanufacturer. The ligation product was packaged in a lambda packagingextract also purchased from Stratagene, and used to infect E. coli BB4cells.

Example 30

Isolation of Pollen-Specific cDNA Clones

The maize pollen cDNA library was probed using the syntheticoligonucleotides probes specific for the 13 kD protein gene, asdescribed in Sambrook et al. Briefly, about 100,000 phage plaques of thepollen cDNA library were plated and lifted to nitrocellulose filters.The filters were probed using oligonucleotides #51 and #58 which hadbeen 32P end-labeled using polynucleotide kinase. The probes werehybridized to the filters at low stringency (50 degrees C. in 1M NaCl,10% dextran sulfate, 0.5% SDS), washed 30 minutes at room temperatureand then 30 minutes at 45 degrees C. in 6×SSC, 0.1% SDS, and exposed toX-ray film to identify positive clones. Putative clones were purifiedthrough four rounds of plaque hybridization. Three classes of cDNAclones were isolated. Type I contained EcoRI fragments of 0.2 kb and 1.8kb. Type II contained EcoRI fragments of 0.6 kb, 0.5 kb and 1.0 kb, andType III contained an EcoRI fragment of 2.3 kb.

Example 31

Characterization of Pollen-Specific cDNA Clones

The EcoRI fragments of the Type II cDNA clone were subcloned into theplasmid vector pBluescript SK+, purchased from Stratagene. See FIG. 29.The 0.6 kb fragment in pBluescript was named II-.6, the 0.5 kb fragmentin pBluescript was named II-.5 (later renamed pCIB3169) and the 1.0 kbfragment in pBluescript was named II-1.0 (later renamed pCIB3168). Aswill be described below, the 0.5 kb and 1.0 kb fragments encode themaize pollen-specific CDPK gene. RNA from anthers, pollen, leaf, rootand silk was denatured with glyoxal, electrophoresed on a 1% agarosegel, transferred to nitrocellulose, and probed separately with the threeEcoRI fragments that had been labeled with 32P by random primerextension as described in Sambrook et al, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press: New York (1989).The blots were exposed to X-ray film, and an mRNA band of approximately1.5 kb was identified with the 0.6 kb fragment probe, while the 0.5 and1.0 kb fragments hybridized to an approximately 2.0 kb mRNA. In allcases hybridization was only seen in the pollen RNA lane, with theexception that the 0.6 kb fragment showed a slight signal in anthermRNA. The conclusion from these data was that the original cDNA clonewas a fusion cDNA molecules derived from two different mRNAs. The 0.6 kbfragment was a partial cDNA of a 1.5 kb pollen-specific mRNA, and thismRNA encodes the peptides LysC 49 and N-terminus. The 1.0 and 0.5 kbfragments comprise a partial cDNA of a 2.0 kb pollen-specific mRNAunrelated to the peptides and oligonucleotide probes used for probes.This conclusion was verified when the fragments were sequenced using thedideoxy chain termination method as described in Sambrook et al. ThecDNA sequence is shown in FIG. 30 (SEQ ID NO: 20).

Example 32

Determination of Specificity of mRNA Expression

To determine if the 2.0 kb RNA represented by cDNA clones pCIB3169 andpCIB3168 were present only in pollen, total RNA was isolated from maize211D roots, leaves, pollen, anthers or silks. The RNAs were denaturedwith glyoxal, electrophoresed on a 1% agarose gel, transferred tonitrocellulose, and probed with 32P-labeled EcoRI insert from plasmidpCIB3168 or pCIB3169, all using standard techniques as described inSambrook et al, Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press: New York (1989). Exposure of this blot tophotographic film demonstrates that the gene represented by these twoclones is only transcriptionally active in the pollen (FIG. 31).

Identification of a Pollen-Specific Promoter Example 33

Construction of a Maize Genomic DNA Library

Genomic DNA from maize line 211D young shoots was isolated as describedin Shure et 1, Cell 35:225-233 (1983). The DNA was provided toStratagene, where a genomic DNA library was constructed by cloningSau3AI partially digested DNA into Stratagene's Lambda Dash cloningvector.

Example 34

Genomic DNA Blot Hybridization to Determine Gene Copy Number.

Genomic DNA from maize line 211 D was digested with a number ofrestriction enzymes, the individual digests electrophoresed on anagarose gel, transferred to nitrocellulose, and probed with 32P-labeledEcoRI insert from plasmid pCIB3168 (1.0 kb fragment), pCIB3169 (0.5 kbfragment) or clone II-.6 using standard techniques described in Sambrooket al. More than 10 bands were detected by the II-.6 probe on mostdigests, indicating that this cDNA is derived from a large, multigenefamily. Probing with the 1.0 kb fragment detected from 3 to 6 bands, andprobing with the 0.5 kb fragment detected only from 1 to 3 bands whichwere a subset of those detected by the 1.0 kb fragment. Due to thesmaller gene family size detected by the 1.0 kb and 0.5 kb fragments, itwas decided to attempt to isolate the genomic clone corresponding tothem.

Example 35

Isolation of a Pollen-Specific Genomic Clone

The Stratagene maize 211D genomic library was screened by probing plaquelifts with 32P labeled inserts from plasmid pCIB3168 (1.0 kb fragment)and pCIB3169 (0.5 kb fragment) using standard procedures as described inthe Stratagene manual accompanying the library. Using this strategy,Lambda clone MG14 was isolated, and it hybridized to both probes. The9.0 kb BamHI fragment of MG14, which also hybridized to both probes, wassubcloned into the BamHI site of pBluescript SK+ to create plasmidpCIB379. 1800 bp of pCIB379, in the region corresponding to the cDNAsequence, was sequenced as described above. Comparison of the cDNA andgenomic sequences showed only 91% identity. pCIB379 insert represents arelated pollen-specific gene.

A second maize 211D genomic library was constructed in the vector lambdaGEM-11, purchased from Promega, using the procedures described in thePromega manual. Screening this un-amplified library as above yieldedclone GEM11-1, which hybridized to both 0.5 and 1.0 kb probes. The 20 kbHindIII fragment of GEM11-1, which also hybridized to both probes, wassubeloned into the HindIII site of pBluescript SK+ to yield pCIB3166.The DNA sequence of 41. kb of pCIB3166 was determined (FIG. 35; SEQ IDNO: 26) and after accounting for six introns in the genomic clone, was100% identical to the cDNA sequence of pCIB3168 and pCIB3169. Comparisonof the pCIB3166 sequence to the Genbank/EMBL database revealed that the5' portion, through the 3 exon, was 34.6% identical to ratcalmodulin-dependent protein kinase II at the amino acid level (FIG.32), while the fourth through seventh exons were 39.4% identical tohuman calmodulin. See FIG. 33. No other pollen-specific kinase has beendescribed, and at the time this a protein combining kinase andcalmodulin domains was unknown. Subsequently, Harper et al., Science252:951-954 (1991) have disclosed the cDNA sequence of a similar proteinfrom soybean, although this gene is not pollen-specific in expression.Comparison of the soybean calcium-Dependent Protein Kinase (CDPK) andthe maize pollen CDPK reveals 38% identity at the amino acid level. SeeFIG. 34.

Example 36

Identification of the Promoter's Transcriptional Start Site by PrimerExtension

Oligonucleotide PE51, with the following sequence was synthesized as aprimer.

5'-TGGCCCATGGCTGCGGCGGGGAACGAGTGCGGC-3' (SEQ ID NO: 81)

Primer extension analysis was carried out on polyA+pollen mRNA asdescribed in Metraux et al., PNAS USA 86:896-890 (1989). Thetranscription initiation site was determined to be between bases 1415and 1425 on the partial sequence of pCIB3166 shown in FIG. 35.

Testing Promoter Function in Transgenic Plants Example 37

Construction of Promoter Vectors for Plant Transformation

To demonstrate that the pollen CDPK promoter can drive expression of alinked gene in transgenic plants, a gene fusion of the pollen CDPKpromoter to the Beta-glucuronidase gene of E. coli was constructed asfollows. The 10 kb BamHI fragment from lambda GEM11-1 containing thefirst exon and part of the first intron of the pollen CDPK gene plus 9kb upstream of the gene was subcloned into the BamHI site of pBluescriptSK+ to create plasmid pCIB3167. The 2.3 kb BamHI-HindIII fragment frompCIB3167 was subcloned into the BamHI and HindIII sites of pBluescriptSK+ to create plasmid pSK105. The pSK105 was digested with AvaI andHindIII, and the 1.75 kb HindIII-AvaI fragment was isolated on anagarose gel. A PCR reaction was run under standard conditions asdescribed in Sambrook et al. using intact pSK105 as a template and thefollowing primers:

#42: 5'-AGCGGTCGACCTGCAGGCATGCGATCTGCACCTCCCGCCG-3' (SEQ ID NO: 82)

#43: 5'-ATGGGCAAGGAGCTCGGG-3 (SEQ ID NO: 83)

The PCR reaction products were digested with Aval and SalI and theresulting fragment isolated on an agarose gel. pBluescript SK+ wasdigested with HindIII and SalI. The 1.75 kb HindIII-AvaI fragment, PCRderived AvaI-SalI fragment, and pBluescript vector with HindIII and SalIends were ligated in a three way ligation to create plasmid pSK 110.

A fusion of the promoter fragment in pSK110 to the Beta-glucuronidase(GUS) gene was created by digesting pSK 110 with HindIII and SalI,isolating the 1.9 kb fragment on an agarose gel and ligating it intoHindIII and SalI sites of pCIB3054, to create plasmid pKL2, a plasmidderived from pUC19 containing the GUS gene followed by plant intron fromthe maize PEPC gene and a polyA signal from cauliflower mosaic virus.This promoter fusion was inactive in plants, probably due to thepresence of out of frame ATG codons in the leader sequence preceding theGUS gene ATG.

A function fusion of the promoter was created by digesting pKL2 withXbaI and SalI to remove the previous fusion junction. A new fusionjunction was produced in a PCR reaction using pSK105 as a template andthe following primers:

#SK50: 5'-CCCTTCAAAATCTAGAAACCT-3' (SEQ ID NO: 84)

#SK49: 5'-TAATGTCGACGAACGGCGAGAGATGGA-3' (SEQ ID NO: 85)

The PCR product was digested with XbaI and SalI and purified on anagarose gel. The purified fragment was ligated into the XbaI and SalIsites of pKL2 to created plasmid pCIB3171. This plasmid contains afunctional fusion of pollen CDPK promoter and GUS which directsexpression the GUS gene exclusively in pollen.

To create a vector containing the pollen CDPK promoter-GUS fusionsuitable for use in Agrobacterium tumefaciens-mediated planttransformation, the fusion gene was isolated from pCIB3171 by digestionwith HindIII and SalI. The resulting fragment was ligated into theHindIII and SalI sites of pBI101 (purchased from Clontech) to createplasmid pCIB3175.

Example 38

Production of Transgenic Plants

pCIB3175 was transformed into Agrobacterium tumefaciens containing thehelper plasmid pCIB542, and the resulting culture used to transform leafdisks from tobacco shoot tip cultures as described by Horsch et al.,Science 227:1229-1231 (1985) except that nurse cultures were omitted andselection was on 100 mg/l kanamycin. Transgenic plants were regeneratedand verified for presence of the transgene by PCR.

Example 39

GUS Gene Expression Analysis

Pollen from primary transformants and their progeny were analyzedhistochemically for expression of the GUS gene as described by Guerreroet al., Mol. Gen. Genet. 224:161-168 (1990). The percentage of pollengrains expressing the GUS gene, as demonstrated by blue staining in theX-gluc buffer, is shown in the table below.

    ______________________________________                                        Plant Number   % Blue Pollen                                                  ______________________________________                                        PP1-51         28%                                                            PP1-54         54%                                                            PP1-55         none                                                           PP1-61         very few                                                       PP1-63         51%                                                            PP1-67         15%                                                            PP1-80         10%                                                            PP1-83         12%                                                            ______________________________________                                    

Primary transformants in which a single pollen CDPK promoter-GUS genewas integrated would produce a maximum 50% GUS positive pollen due tosegregation of the single gene. Flouometric GUS assays were done onpollen, stem, root, leaf and pistil tissue of selected plants todemonstrate the specificity of pollen CDPK promoter expression. Assayswere performed as described in Jefferson, Plant Mol. Biol.14:995-1006(1990), and GUS activity values are expressed as nmoles MU/ugprotein/minute.

    ______________________________________                                                        Untransformed                                                 Plant                     Net      Plant                                      number                                                                              Tissue  GUS Activity                                                                              GUS Activity                                                                           GUS Activity                               ______________________________________                                        PP1-51                                                                              stem    0.01        0.02     0                                                leaf    0           0        0                                                root    0.15        0.10     0.05                                             pistil  0.02        0.01     0.01                                             pollen  0.24        0.02     0.22                                       PP1-54                                                                              stem    0.01        0.02     0                                                leaf    0           0        0                                                root    0.13        0.1      0.03                                             pistil  0.01        0.01     0                                                pollen  0.60        0.02     0.58                                       PP1-63                                                                              stem    0.01        0.02     0                                                leaf    0           0        0                                                root    0.07        0.1      0                                                pistil  0.01        0.01     0                                                pollen  0.57        0.02     0.55                                       ______________________________________                                    

Examples 40-50 are directed primarily to the preparation of chimericconstructs, i.e. recombinant DNA molecules, containing constitutive,tissue-preferred, or tissue-specific promoters operably linked to aninstant B.t. gene, insertion of same into vectors, production oftransgenic platns containing the vectors, and analysis of expressionlevels of B.t. proteins of the transgenic plants.

Example 40

CONSTRUCTION OF MAIZE OPTIMIZED BT TRANSFORMATION VECTORS

To demonstrate the effectiveness of the synthetic Bt cryIA(b) gene inmaize, the PepC and pith specific promoters are fused to the syntheticBt cryIA(b) gene using PCR. Oligomers designed for the PCR fusions were:

(PEPC)

KE99A28=5'-TGCGGTTACC GCCGATCACATG-3' (SEQ ID NO: 86)

KE97A28=5'-GCGGTACCGC GTCGACGCGG ATCCCGCGGC GGGAAGCTAAG-3' (SEQ ID NO:87)

(PITH)

KE100A28=5'-GTCGTCGACC GCAACA-3' (SEQ ID NO: 88)

KE98A28=5'-GCGGTACCGC GTTAACGCGG ATCCTGTCCG ACACCGGAC-3' (SEQ ID NO: 89)

KE104A28=5'-GATGTCGTCG ACCGCAACAC-3' (SEQ ID NO: 90)

KE103A28=5'-GCGGTACCGC GGATCCTGTC CGACACCGGA CGGCT-3' (SEQ ID NO: 91)

PCR primers are designed to replace the Nco I sites in the 5'untranslated leader region of each of these tissue specific genes(containing ATG translational start sites) with Bam HI sites tofacilitate cloning of the synthetic cryIA(b) gene into this Bam HI site.Subsequent construction of vectors containing the tissue specificpromoters fused to the synthetic cryIA(b) gene and also containing the35S:PAT:35S marker gene involves several intermediate constructs.

1. pCIB4406 (35S:synthetic-cryIA(b):pepC ivs#9:35S)

pCIB4406 contains the 2 Kb Bam HI\Cla I synthetic cryIA(b) gene fusedwith the CaMV 35S promoter (Rothstein et al., Gene 53:153-161 (1987)).The gene also contains intron #6 derived from the maize PEP carboxylasegene (ivs#9) in the 3' untranslated region of the gene, which uses theCaMV 3' end. (PNAS USA, 83:2884-2888 (1986), Hudspeth et al., PlantMolecular Biology, 12: 579-589 (1989)). pCIB4406 is ligated andtransformed into the "SURE" strain of E. coli cells (Stratagene, LaJolla, Calif.) as described above. One mutation is found in pCIB4406'scryIA(b) gene at amino acid #436 which resulted in the desired the beingchanged to a Leu. pCIB4406 is fully active against European corn borerwhen tested in insect bioassays and produces a CryIA(b) protein of theexpected size as determined by western blot analysis.

2. pCIB4407 (35S:synthetic-cryIA(b):pepC ivs#9:35S +35S:PAT:35S)

pCIB4407 is made from an approximately 4 Kb Hind III\Eco RI fragmentcontaining the 35S:PAT:35S gene, and the 3.1 Kb\Hind HIII\Eco RI35S:synthetic-cryLA(b):35S gene from pCIB4406. pCIB4407 is ligated andtransformed into "SURE", DH5alpha, and HB101 strains of E. coli usingstandard procedures (Sambrook et al.). The synthetic cryIA(b) gene hasthe same properties as its precursor pCIB4406.

3. pCIB4416 (35S:synthetic-cryIA(b):pepC ivs#9:35S +35S:PAT:35S+35S:Adhintron:GUS:35S.)

pCIB4407 is cut with Eco RI and treated with calf intestinal alkalinephosphatase (CIP) under standard conditions (Sambrook et al.) to producean about 7.2 Kb fragment that is ligated with a 3.4 Kb Eco RI35S:Adh\GUS:35S fragment to produce pCIB4416. Ligations andtransformations into "SURE" cells is as described above. The syntheticcryIA(b) gene in pCIB4416 has the same properties as the gene inpCIB4406.

4. pCIB4418 (35S:synthetic-cryIA(b):pepC ivs#9:35S)

pCIB4406 is digested with Apa I and Bam HI and treated with CIP.pCIB4406 is digested with Bam HI and Nsp I. pBS123#13 is digested withNsp I and Apa I. A three-way ligation is made consisting of a 4.3 Kb ApaI\Bam HI fragment from pCIB4406, a 1.3 Kb Bam HI\Nsp I fragment frompCIB4406, and a 170 bp Nsp I\Apa I fragment from pBS 123#13 to formpCIB4418. The host E. coli strain for pCIB4418 is HB 101.

5. pCIB4419 (35S:synthetic-cryIA(b):pepC ivs#9:35S+35S:PAT:35S+35S:Adhintron:GUS:35S.)

pCIB4416 and pCIB4418 are digested with Bst E II and Eco NI andfragments of pClB4416 are treated with CIP. A 9.1 Kb fragment frompCIB4416 ligated to a 1.4 Kb fragment from pCIB4418 to form pCIB4419.pCIB4419 transformed in HB101 competent E. coli cells demonstrates fullactivity in insect bioassays against European corn borer.

6. pCIB4420 (Pith:synthetic-cryIA(b):PEPC ivs#9:35S+35S:PAT:35S)

Intermediate constructs in making pCIB4420 are pBTin1, pBtin2, p4420Aand pBtin3. pBtin1 (pith promoter:second half of the synthetic Btgene+35S:PAT:35S) is made by ligating the 2.1 Kb Xba I\Nco I pithpromoter fragment from plasmid pith(3-1) with a 5.2 Kb Xba I\Nco Ifragment from pCIB4407. pBtin2 is an intermediate construct containingthe pith promoter modified with a 210 bp PCR fragment made using primersKE100A28 and KE98A28 listed above. The PCR reaction mix containsapproximately 100 ng of a 2.1 Kb Bam HI\Nco I pith promoter fragmentwith 100 pmol of each oligomer, 200 nM of each dNTP, 1×buffer (Cetus)and 2.5 units of thermal stable polymerase. Since the Tm is relativelylow (between 40° and 50° C.), PCR reactions are run with the followingparameters:

denaturation cycle: 94° C. for 1 minute

annealing cycle: 37° C. for 1 minute

extension cycle: 72° C. for 45 seconds (+3 seconds per cycle)

number of cycles: 25

PCR reactions are treated with proteinase K as described above prior tocutting with Sal I\Kpn I followed by phenol\chloroform extraction andethanol precipitation as described above. The 210 bp fragment ispurified on a 2% Nusieve gel and extracted from the gel usingMillipore's filter units. The 210 bp Sal I\Kpn I fragment is ligated tothe 4.9 Kb Sal I\Kpn I fragment from pith(3-1) to make pBtin2. p4420A(pith:synthetic-Bt:Pep intron:35S+35S:PAT:35S) is made with a three-wayligation consisting of a 700 bp Nsi I\Bam HI fragment from pBtin2, a 1.8Kb Bam HI\Bst E II fragment from pCIB4418, and a 5.9 Kb Bst E II\Nsi Ifragment from pBtin 1. After p4420A is made three mutations arediscovered in pBtin2. A second PCR fragment is made to modify the Nco Isite in the pith leader using primers KE104A28 and KE103A28 with Tmvalues around 65° C. The PCR reaction mix is identical to that listedabove with the addition of glycerol to 20% to reduce mutations in G+Crich areas (Henry et al., Plant Molecular Biology Reporter 9(2):139-144,1991). PCR parameters are as follows:

File I: 94° C.: 3 minutes, 1 cycle

File II: 60° C.: 1 minute

94° C.: 1 minute

25 cycles

File III: 72° C.: 5 minutes, 1 cycle

PCR reactions are treated as above and cut with restrictionendonucleases Sal I and Kpn I. The 210 bp Sal I\Kpn I PCR (glycerol inthe reaction) fragment is ligated to the 4.9 Kb Sal I\Kpn I fragmentfrom plasmid pith(3-1) to make pBtin3. Sequence data on pBtin3-G#1 showsthis PCR generated fragment to be correct.

pBtin3-G#1 is used to make pCIB4420 (also called p4420B "G#6"). pCIB4420is constructed with a three-way ligation using the 700 bp Nsi I\Bam HIfragment from pBtin3-G#1, a 1.8 Kb Bam HI\Bst E II fragment frompCIB4418, and a 5.9 Kb Bst E II\Nsi I fragment from pBtin1. pCIB4420 isused in mesophyll protoplast experiments and demonstrates full activityof the synthetic cryIA(b) gene against European corn borer.

7. pCIB4413 (PEPC:synthetic-Bt (Phe mutation):PEPC intron:35S.) A fusionfragment is generated by PCR using primers KE99A28 and KE97A28 with a2.3 KB Hind III\Sal I template from pGUS4.5. The PCR mix contains thesame concentration of primers, template, dNTPs, salts, and thermalstable polymerase as described above. PCR reaction parameters are:

denaturation cycle: 94° C. for 1 minute

annealing cycle: 55° C. for 1 minute

extension cycle: 72° C. for 45 seconds (+3 seconds per cycle)

number of cycles: 30

After completion, PCR reactions are treated with proteinase K followedby phenol\chloroform extraction and ethanol precipitation as describedabove prior to cutting with restriction endonucleases Bam HI and Bst EII.

pCIB4413 is made with a three-way ligation using the 210 bp Bam HI\Bst EII PCR fragment, a 4.7 Kb Bam HI\Hind III fragment from pCIB4406, and a2.2 Kb Hind III\Bst E II fragment from pGUS4.5.

8. pCIB4421 (PEPC:synthetic-cryIA(b):PEPC intron:35S.) pCIB4421 is madeto replace the synthetic cryIA(b) gene containing the Phe mutation inpCIB4413 with the synthetic cryIA(b) gene from pCIB4419. pCIB4421 ismade by ligating a 5.2 Kb Bam HI\Sac I fragment from pCIB4413 with a 1.9Kb Bam HI\Sac I fragment from pCIB4419.

9. pCIB4423 (PEPC:synthetic-cryIA(b):PepC intron:35S+35S:PAT:35S)

The 2.4 Kb Bam HI\Hind III PEPC promoter fragment from pCIB4421 isligated to the 6.2 Kb Bam HI\Hind III fragment in pCIB4420 to makepCIB4423. The Hind III site is deleted by exonucleases in the cloning ofpCIB4423. pCIB4423 contains the synthetic cryIA(b) gene under thecontrol of the PEPC promoter, and the PAT gene under the control of the35S promoter.

10. Synthetic cryIA(b) gene in Agrobacterium strains:

Agrobacterium strains made with the synthetic cryIA(b) gene allowtransfer of this gene in a range of dicotyledenous plants. Agrobacteriumvector pCIB4417 contains the 3.3 Kb Hind III\Eco RI35S:synthetic-CryIA(b):PepC:ivs#9:35S fragment from pCIB4406 (Phemutation) ligated to the 14 Kb Hind III\Eco RI fragment from pBI101(Clontech). Using electroporation, pCIB4417 is transferred into the A.tumefaciens strain LBA4404 (Diethard et al., Nucleic Acids Research, Vol17:#16:6747, 1989.).

200 ng of pCIB4417 and 40 ul of thawed on ice LBA4404 competent cell areelectroporated in a pre-cooled 0.2 cm electroporation cuvette (Bio-RadLaboratories Ltd.). Using Gene Pulser-TM with the Pulse Controller unit(Bio-Rad), an electric pulse is applied immediately with the voltage setat 2.5 kV, and the capacity set at 25 uF. After the pulse, cells areimmediately transferred to 1 ml of YEB medium and shaken at 27° C. for 3hours before plating 10 ul on ABmin:Km50 plates. After incubating at 28°C. for approximately 60 hours colonies are selected for miniscreenpreparation to do restriction enzyme analysis. The final Agrobacteriumstrain is called pCIB4417:LBA4404.

Example 41

ELISA ANALYSIS OF TRANSFORMED MAIZE PROTOPLASTS

The presence of the cryIA(b) toxin protein is detected by utilizingenzyme-linked immunosorbent assay (ELISA). ELISAS are very sensitive,specific assays for antigenic material. ELISA assays are useful todetermine the expression of polypeptide gene products. Antiserum forthese assays is produced in response to immunizing rabbits withgradient-purified Bt crystals (Ang et al., Applied Environ. Microbiol.,36:625-626 (1978)) solubilized with sodium dodecyl sulfate. ELISAanalysis of extracts from transiently transformed maize cells is carriedout using standard procedures (see for example Harlow, E., and Lane, D.in "Antibodies: A Laboratory Manual", Cold Spring Harbor LaboratoryPress, 1988). ELISA techniques are further described in Clark et al.,Methods in Enzymology, 118:742-766 (1986); and Bradford, Anal. Biochem.,72:248 (1976). Thus, these procedures are well-known to those skilled inthe art. The disclosure of these references is hereby incorporatedherein by reference.

ELISA assays are performed to detect the production of CryIA(b) proteinin maize protoplasts. Protein produced is reported below as ng of Bt permg total protein (ng Bt/mg). Each construct was tested twice.

    ______________________________________                                        pCIB3069        No detectable Bt (both tests)                                 pCIB4407        21,900 ng Bt/mg total protein,                                                21,000 ng Bt/mg total protein                                 ______________________________________                                    

The transformed maize cells produce high levels, on the order ofapproximately 20,000 ng of Bt CryIA(b) protein per mg total solubleprotein, of the Bt IP when transformed with the maize optimized Bt gene.The level of detection of these ELISA based assays is about 1 to 5 ngCryIA(b) protein per mg protein. Therefore, the maize optimized Bt geneproduces as much as approximately a 20,000 fold increase in expressionof this protein in maize cells.

Example 42

ASSAY OF EXTRACT FROM TRANSFORMED PROTOPLASTS FOR INSECTICIDAL ACTIVITYAGAINST EUROPEAN CORN BORER

Western blot analysis is also performed using extracts obtained frommaize cells which had been transiently transformed with DNA to expressthe maize optimized gene. When examined by western blots, this proteinappears identical with the protein produced in E. coli. In contrast, asdemonstrated in Example 6 above, no detectable Bt cryIA(b) insecticidalprotein is produced by maize cells transformed with comparable vectorsattempting to express the native Bt derived coding region.

Qualitative insect toxicity testing can be carried out using harvestedprotoplasts. Suspensions are prepared for each replicate tested in allbioassays. A replicate is considered positive if it causes significantlyhigher mortality than the controls. For example, replicates are testedfor their activity against insects in the order Lepidoptera by using theEuropean corn borer, Ostrinia nubilalis. One-hundred μl of a protoplastsuspension in 0.1% Triton X-100 is pipetted onto the surface ofartificial Black cutworm diet, (Bioserv, Inc., Frenchtown, N.J.; F9240)in 50 mm×10 mm snap-cap petri dishes. After air drying 10 neonatallarvae are added to each plate. Mortality is recorded after about 4days. When this protein is fed to European corn borers, it produces 100%mortality.

Example 43

EXPRESSION OF SYNTHETIC BT IN MAIZE MESOPHYLL PROTOPLASTS

The general procedure for the isolation of corn mesophyll protoplasts isadapted from Sheen et al., The Plant Cell, 2:1027-1038 (1990). Theprotoplast transformation system used in Sheen et al. is modified byusing PEG mediated transformation, rather than electroporation. Thatprocedure, as well as changes made in the isolation procedure, isdescribed below. Maize Mesophyll Protoplast Isolation/Fransformation

1. Sterilize and germinate corn seeds for leaf material. Seedlings aregrown in the light at 25° C.

2. Surface sterilize leaf pieces of 10-12 day old seedlings with 5%Clorox for 5 minutes followed by several washes with sterile distilledwater.

3. Aliquot enzyme solution (see recipe below); 25 ml/dish (100×25 mmpetri dish).

4. Remove any excess water from leaves and place 6-8 2 inch pieces ineach dish of enzyme. 14 plates are usually set up with the leaf materialfrom about 100 seedlings.

5. Cut leaves in longitudinal strips as thin as possible (2-5 mm).

6. Shake slowly at 25° C. for 6.5 to 7 hours. Cover plates so thatincubation takes place in the dark.

7. Before filtering protoplasts, wash 100 um sieves with 10 ml 0.6 Mmannitol. Pipet protoplasts slowly through sieves. Wash plates with 0.6M mannitol to gather any protoplasts left in the dishes.

8. Pipet filtered liquid carefully into 50 ml sterile tubes. Add equalvolumes of 0.6 M mannitol to dilute.

9. Spin for 10 minutes at 1000 rpm/500 g in table-top centrifuge(Beckman Model TJ-6).

10.Remove enzyme solution and discard. Resuspend pellets carefully in 5ml mannitol. Pool several pellets. Bring volume to 50 ml with 0.6 Mmannitol and spin.

11. Resuspend to a known volume (50 ml) and count.

12. After counting and pelleting, resuspend protoplasts at 2 million/mlin resuspending buffer (recipe below). Allow ppts to incubate in theresuspending buffer for at least 30 min before transformation.

Transformation:

1. Aliquot plasmids to tubes (Fisherbrand polystyrene 17×100 mm Snap Capculture tubes); at least three replicates per treatment; use equimolaramounts of plasmids so that equal gene copy numbers are compared.

2. Add 0.5 ml protoplasts and 0.5 ml 40% PEG made with 0.6 M mannitol.

3. Shake gently to mix and incubate at 25° C. for 30 min.

4. Add protoplast culture media at 5 min intervals: 1,2,5 ml

5. Spin for 10 min at 1000 rpm/500 g.

6. Remove liquid from pellet and resuspend in 1 ml culture media (BMVmedia)

7. Incubate overnight at 25° C. in the dark.

Recipes:

Enzyme Solution

0.6 M mannitol

10 mM MES, pH 5.7

1 mM CaCL₂

1 mM MgCl₂

0.1% BSA

filter-sterilize

To this solution, add the following enzymes:

1% Cellulase RS, and 0.1% Macerozyme R10

Wash Buffer: 0.6 M mannitol, filter-sterilize

Resuspending Buffer: 0.6 M mannitol, 20 mM KCl, filter-sterilize

Culture Media: BMV media recipe from:

Okuno et al., Phytopathology 67:610-615 (1977).

0.6 M mannitol

4 mM MES, pH5.7

0.2 mM KH₂ PO₄

1 mM KNO₃

1 mM MgSO₄

10 mM CaCI₂

1×K3 micronutrients

filter-sterilize

ELISA analysis of transformed protoplasts is done one day aftertransformation. ELISA's are done as previously described. The followingthree experiments are done with maize inbred line 211D. Of course, otherlines of maize may be used. 50 ug of plasmid pCIB4419 and equimolaramounts of other plasmids are used. Total soluble protein is determinedusing the BioRad protein assay. (Bradford, Anal.Biochem, 72:248 (1976).

Transformation Experiment:

Constructs tested:

1. pCIB4419 (Construct contains synthetic Bt under control of CaMV 35Spromoter and 35S/PAT and 35S/GUS marker genes)

2. pCIB4420 (Construct contains synthetic Bt under control of Pithpromoter and PAT marker gene)

3. pCIB4421 (Construct contains synthetic Bt under control of PEPCpromoter)

4. pCIB4423 (Construct contains synthetic Bt under control of PEPCpromoter and PAT marker gene) (PEPC:synthetic-cryIA(b):PepCintron:35S+35S:PAT:35S)

In the following experiments, 10 or 11 day old 211D seedlings areanalyzed for production of the Bt CryIA(b) protein in the Biorad proteinassay:

Experiment 1 (11 day seedlings):

    ______________________________________                                        pCIB4419       15,000 ± 3,000 ng Bt/mg protein                             pCIB4420       280 ± 65 ng Bt/mg protein                                   pCIB4421       9,000 ± 800 ng Bt/mg protein                                ______________________________________                                    

Experiment 2 (10 day seedlings):

    ______________________________________                                        pCIB4419        5,000 ± 270 ng Bt/mg protein                               pCIB4420        80 ± 14 ng Bt/mg protein                                   pCIB4421        1,600 ± 220 ng Bt/mg protein                               ______________________________________                                    

Experiment 3 (11 day seedlings):

    ______________________________________                                        pCIB4419       21,500 ± 1,800 ng Bt/mg protein                             pCIB4420       260 ± 50 ng Bt/mg protein                                   pCIB4421       11,900 ± 4,000 ng Bt/mg protein                             pCIB4423       7,200 ± 3,400 ng Bt/mg protein                              ______________________________________                                    

The above experiments confirm that both the CaMV 35S and PEPC promotersexpress the synthetic Bt CryIA(b) protein at very high levels. The pithpromoter, while less efficient, is also effective for the expression ofsynthetic CryIA(b) protein.

Example 44

STABLE EXPRESSION OF SYNTHETIC BT IN LETTUCE

The synthetic Bt gene in the Agrobacterium vector pCIB4417 istransformed into Lactuca sativa cv. Redprize (lettuce). Thetransformation procedure used is described in Enomoto et al., Plant CellReports, 9:6-9 (1990).

Transformation procedure:

Lettuce seeds are suface sterilized in 5% Clorox for 5 minutes followedby several washes in sterile distilled water. Surface-sterilized seedsare plated on half strength MS media (Murashige and Skoog, Physiol.Plant. 15:473-497 (1962)). Cotyledons of 6-day-old Redprize seedlings,grown under illumination of 3,000 l×16 hr at 25° C., are used as theexplants for Agrobacterium infection. The base and tip of each cotyledonare removed with a scalpel. The explants are soaked for 10 minutes inthe bacterial solution which have been cultured for 48 hours in ABminimal media with the apropriate antibiotics at 28° C. After blottingexcess bacterial solution on sterile filter paper, the explants areplated on MS media (0.1 mg/l BA and 0.1 mg/l NAA) for 2 days. Explantsare then transferred to selective media containing 500 mg/lcarbenicillin and 50 mg/l kanamycin. The explants are subcultured tofresh media weekly. The growth chamber conditions are 16 hour 2,000l×light at 25° C. After approximately 4 weeks, an ELISA is done onhealthy looking callus from each of four plates being subcultured. TheELISA procedure is the same as described above for protoplasts; solubleprotein is again determined by the Biorad assay described above.

Results:

    ______________________________________                                        pCIB3021 (kan control)                                                                           0                                                          pCIB4417 (plate 1) 0                                                          pCIB4417 (plate 2) 505 ng Bt/mg protein                                       pCIB4417 (plate 3) 45 ng Bt/mg protein                                        pCIB4417 (plate 4) 1,200 ng Bt/mg protein                                     ______________________________________                                    

This example demonstrates that dicot plants can also show increasedexpression of the optimized insecticidal gene.

Example 45

Construction of pCIB4429.

pCIB4429 contains a preferred maize pollen-specific promoter fused withthe maize optimized cryIA(b) gene. The pollen-specific maize promoterused in this construct was obtained from the plasmid pKL2, described inExample 37. The maize optimized cryIA(b) gene was obtained from plasmidpCIB4418, also described in Example 37.

pKL2 is a plasmid that contains a preferred maize pollen-specificpromoter fused with the E. coli beta-glucuronidase gene. It wasconstructed from plasmids pSKl 10 and pCIB3054. pSK110 contains thepollen specific maize promoter. pCIB3054, a pUC19 derivative, containsthe E. coli beta-glucuronidase (GUS) gene fused with the cauliflowermosaic virus (CaMV) 35S promoter. It's construction is describedelsewhere in this application. This promoter can be removed from thisplasmid by cutting with SalI/HindIII to yield a fragment containing theGUS gene, a bacterial ampicillin resistance gene and a ColEI origin ofreplication. A second fragment contains the CaMV 35S promoter.

pCIB3054 was cut with the restriction enzymes SalI and HindIII, usingstandard conditions, for 2 hours at room temperature. The reaction wasthen extracted with phenol/chloroform using standard conditions and theDNA recovered by ethanol precipitation using standard conditions. Therecovered DNA was resuspended in buffer appropriate for reaction withcalf intestinal alkaline phosphatase (CIP) and reacted with 2.5 units ofCIP at 37° C. overnight. After the CIP reaction, the DNA was purified onan agarose gel using standard conditions described elsewhere in thisapplication. pSK10 was cut with SalI/HindIII under standard conditionsfor 2 hours at room temperature and the DNA subsequently purified on anagarose gel using standard conditions. The recovered DNA fragments wereligated using standard conditions for two hours at room temperature andsubsequently transformed into competent E. coli strain HB 101 cellsusing standard conditions. Transformants were selected on L-agarcontaining 100 μg ampicillin/ml. Transformants were characterized forthe desired plasmid construct using standard plasmid mini-screenprocedures. The correct construct was named pKL2.

To make pCIB4429, a three way ligation was performed using standardconditions known to those in the art. The three fragments ligated were:

1) a HindIII/(BamHI fragment from pCIB4418, of about 4.7 kb in size,containing the cryIA(b) gene, the bacterial ampicillin resistance gene,and the ColEI origin of replication

2) a HindIII/XbaI fragment from pKL2 of about 1.3 kb in size andcontaining the pollen specific promoter from maize

3) a PCR generated fragment derived from the pollen promoter with aBamHI site introduced downstream from the start of transcription. Thisfragment is approximately 120 bp and has ends cut with the restrictionenzymes XbaI/BamHI.

The PCR fragment was generated using a 100 μl reaction volume andstandard conditions described above. The primers used were:

SK50: 5'-CCC TTC AAA ATC TAG AAA CCT-3' (SEQ ID NO: 84)

KE127: 5'-GCG GAT CCG GCT GCG GCG GGG AAC GA-3' (SEQ ID NO: 92)

The above primers were mixed in a PCR reaction with plasmid pSK105, aplasmid that contains the pollen specific promoter from maize.

After the PCR reaction was complete, 10 μl of the reaction was run on anagarose gel, using standard condition, to make sure the reactionproduced the expected size product. The remaining 90 μl was treated withproteinase K at a final concentration of 50 μg/ml for 30 min. at 37° C.The reaction was then heated at 65° C. for 10 min., thenphenol/chloroform extracted using standard procedures. The DNA wasrecovered from the supernatant by precipitating with two volumes ofethanol using standard conditions. After precipitation, the DNA wasrecovered by centrifuging in a microfuge. The pellet was rinsed one timewith 70% ethanol (as is standard in the art), briefly dried to removeall ethanol, and the pellet resuspended in 17 μl TE buffer. 2 μl of10×restriction enzyme buffer was added as were 0.5 μl BamHI and 0.5 μlXbaI. The DNA was digested for 1 hour at 37° C. to produce a DNAfragment cut with XbaI/BamHI. After digestion with the restrictionenzymes, this fragment was purified on an agarose gel composed of 2%NuSieve (FMC)/1% agarose gel. Millipore filter units were used to elutethe DNA from the agarose using the manufacturer's specifications. Afterelution, the DNA was used in the three-way ligation described above.

After ligation, the DNA was transformed into competent E. coli strain HB101 cells using standard techniques. Transformants were selected onL-agar plates containing ampicillin at 100 μg/ml. Colonies that grewunder selective conditions were characterized for plasmid inserts usingtechniques standard in the art.

Example 46

Construction of pCIB4431, a Vector for Tissue Specific Expression of theSynthetic CryIA(b) Gene in Plants.

pCIB4431 is a vector designed to transform maize. It contains twochimeric Bt endotoxin genes expressible in maize. These genes are thePEP carboxylase promoter/synthetic-cryIA(b) and a pollenpromoter/synthetic-cryLA(b). The PEP carboxylase/crylA(b) gene in thisvector is derived from pCIB4421 described above. The pollen promoter isalso described above. FIG. 20 is a map of plasmid pCIB4431. pCIB4431 wasconstructed via a three part ligation using the about 3.5 Kb Kpn I/HindIII fragment (containing pollen/synthetic-cryIA(b)from pCIB4429, theabout 4.5 Kb Hind III/Eco RI (PEPC/synthetic-cryIA(b) and the about 2.6Kb Kpn I/Eco RI fragment from the vector Bluescript.

Other vectors including the pollen promoter/synthetic CryIA(b) chimericgene include pCIB4428 and pCIB4430. See FIGS. 21 and 22. pCIB4430 alsocontains the PEPC/synthetic-Bt gene described above.

Example 47

Production of Transgenic Maize Plants Containing the Synthetic MaizeOptimized CryIA(b) Gene

The example below utilizes Biolistics to introduce DNA coated particlesinto maize cells, from which transformed plants are generated.

Experiment KC-65

Production of transgenic maize plants expressing the synthetic cryIA(b)gene using a tissue-specific promoter.

Tissue

Immature maize embryos, approximately 1.5-2.5 mm in length, were excisedfrom an ear of genotype 6N615 14-15 days after pollination. The motherplant was grown in the greenhouse. Before excision, the ear was surfacesterilized with 20% Clorox for 20 minutes and rinse 3 times with sterilewater. Individual embryos were plated scutellum side in a 2 cm squarearea, 36 embryos to a plate, on the callus initiation medium, 2DG4+5chloramben medium (N6 major salts, B5 minor salts, MS iron, 2% sucrose,with 5 mg/l chloramben, 20 mg/l glucose, and 10 ml G4 additions(Table 1) added after autoclaving.

                  TABLE 1                                                         ______________________________________                                        G4 Additions                                                                  Ingredient          per liter medium                                          ______________________________________                                        Casein hydrolysate  0.5 gm                                                    Proline             1.38 gm                                                   Nicotinic acid      .2 mg                                                     Pyridoxine-HCl      .2 mg                                                     Thiamine-HCl        .5 mg                                                     Choline-HCl         .1 mg                                                     Riboflavin          .05 mg                                                    Biotin              .1 mg                                                     Folic acid          .05 mg                                                    Ca pantothenate     .1 mg                                                     p-aminobenzoic acid .05 mg                                                    B12                 .136 μg                                                ______________________________________                                    

Bombardment

Tissue was bombarded using the PDS-1000He Biolistics device. The tissuewas placed on the shelf 8 cm below the stopping screen shelf. The tissuewas shot one time with the DNA/gold microcarrier solution, 10 μl driedonto the macrocarrier. The stopping screen used was hand punched at ABRUusing 10×10 stainless steel mesh screen. Rupture discs of 1550 psi valuewere used. After bombardment, the embryos were cultured in the dark at25° C.

Preparation of DNA for Delivery

The microcarrier was prepared essentially according to the instructionssupplied with the Biolistic device. While vortexing 50 μl 1.0 μgoldmicrocarrier, added 5 μl pCIB4431 (1.23 μg/μl) (#898)+2 μl pCIB30640.895 μg/μl) (#456) followed by 50 μl 2.5 M CaCl₂, then 20 μl 0.1 Mspermidine (free base, TC grade). The resulting mixture was vortexed 3minutes and microfuged for 10 sec. The supernatant was removed and theicrocarriers washed 2 times with 250 μl of 100% EtOH (HPLC grade) byvortexing briefly, centrifuging and removing the supernatant. Themicrocarriers are resuspended in 65 μl 100% EtOH.

Callus formation

Embryos were transferred to callus initiation medium with 3 mg/l PPT 1day after bombardment. Embryos were scored for callus initiation at 2and 3 weeks after bombardment. Any responses were transferred to callusmaintenance medium, 2DG4+0.5 2,4-D medium with 3 mg/L PPT. Callusmaintenance medium is N6 major salts, B5 minor salts, MS iron, 2%sucrose, with 0.5 mg/l 2,4-D, 20 mg/l glucose, and 10 ml G4 additionsadded after autoclaving. Embryogenic callus was subcultured every 2weeks to fresh maintenance medium containing 3 mg/L PPT. All callus wasincubated in the dark at 25° C.

The Type I callus formation response was 15%. Every embryo whichproduced callus was cultured as an individual event giving rise to anindividual line.

Regeneration

After 12 weeks on selection, the tissue was removed from callusmaintenance edium with PPT and was placed on regeneration medium.Regeneration medium is 0.25MS3S5BA (0.25 mg/l 2,4 D, 5 mg/l BAP, MSsalts, 3% sucrose) for 2 weeks followed by subculture to MS3S medium forregeneration of plants. After 4 to 10 weeks, plants were removed and putinto GA 7's. Our line KC65 0-6, which became the #176 BT event, produceda total of 38 plants.

Assays

All plants, as they became established in the GA7's, were tested by thechlorophenol red (CR) test for resistance to PPT as described in U.S.patent application Ser. No. 07/759,243, filed Sep. 13, 1991, therelevant portions of which are hereby incorporated herein by reference.This assay utilizes a pH sensitive indicator dye to show which cells aregrowing in the presence of PPT. Cells which grow produce a pH change inthe media and turn the indicator yellow (from red). Plants expressingthe resistance gene to PPT are easily seen in this test. (#176=8positive/30 negative) Plants positive by the CR test were assayed by PCRfor the presence of the synthetic BT gene. (#176=5 positive/2 negative/1dead)

Plants positive by PCR for the syn-BT gene were sent to the phytotron.Once established in the phytotron, they were characterized using insectbioassays and ELISA analysis. Plants were insect bioassayed using astandard European Corn Borer assay (described in Example 5A) in whichsmall pieces of leaf of clipped from a plant and placed in a small petridish with a number of ECB neonate larvae. Plants are typically assayedat a height of about 6 inches. Plants showing 100% mortality to ECB inthis assay are characterized further. ELISA data are shown below.Positive plants are moved to the greenhouse.

Greenhouse/Fertility

Plant number #176-11 was pollinated with wild-type 6N615 pollen. Onetassel ear and one ear shoot were produced. All of the embryos from thetassel ear (11) and 56 kernels from Ear 1 were rescued. 294 kernelsremained on the ear and dried down naturally.

Pollen from #176-11 was outcrossed to various maize genotypes 5N984,5NA89, and 3N961. Embryos have been rescued from all 3 outcrosses(5N984=45; 5NA89=30; 3N961=8). Most of the kernels remained on the earson the plants in the greenhouse and were dried down naturally. DNA wasisolated from plant #176-11 using standard techniques and analysed bySouthern blot analysis. It was found to contain sequences whichhybridize with probes generated from the synthetic cryIA(b) gene andwith a probe generated from the PAT gene. These results showedintegration of these genes into the genome of maize.

Experiment KC-64

Production of transgenic maize plants expressing the synthetic cryIA(b)gene using a constitutive promoter.

Tissue

Immature maize embryos, approximately 1.5-2.5 mm in length, were excisedfrom an ear of genotype 6N615 14-15 days after pollination. The motherplant was grown in the greenhouse. Before excision, the ear was surfacesterilized with 20% Clorox for 20 minutes and rinse 3 times with sterilewater. Individual embryos were plated scutellum side in a 2 cm squarearea, 36 embryos to a plate, on the callus initiation medium, 2DG4+5chloramben medium (N6 major salts, B5 minor salts, MS iron, 2% sucrose,with 5 mg/l chloramben, 20 mg/l glucose, and 10 ml G4 additions Table 1)added after autoclaving.

                  TABLE 1                                                         ______________________________________                                        G4 Additions                                                                  Ingredient          per liter medium                                          ______________________________________                                        Casein hydrolysate  0.5 gm                                                    Proline             1.38 gm                                                   Nicotinic acid      .2 mg                                                     Pyridoxine-HCl      .2 mg                                                     Thiamine-HCl        .5 mg                                                     Choline-HCl         .1 mg                                                     Riboflavin          .05 mg                                                    Biotin              .1 mg                                                     Folic acid          .05 mg                                                    Ca pantothenate     .1 mg                                                     p-aminobenzoic acid .05 mg                                                    B12                 .136 μg                                                ______________________________________                                    

Bombardment

Tissue was bombarded using the PDS-1000He Biolistics device. The tissuewas placed on the shelf 8 cm below the stopping screen shelf. The tissuewas shot one time with the DNA/gold microcarrier solution, 10 μl driedonto the macrocarrier. The stopping screen used was hand punched at ABRUusing 10×10 stainless steel mesh screen. Rupture discs of 1550 psi valuewere used. After bombardment, the embryos were cultured in the dark at25° C.

Preparation of DNA for Delivery

The microcarrier was prepared essentially according to the instructionssupplied with the Biolistic device. While vortexing 50 μl 1.0 μl goldmicrocarrier, added 3.2 μl pCIB4418 (0.85 μg/μl) (#905)+2 μl pCIB30640.895 μg/μl)(#456)+1.6 μl pCIB3007A (1.7 μg/μl) (#152) followed by 50 μl2.5 M CaCl₂, then 20 μl 0.1 M spermidine (free base, TC grade). Theresulting mixture was vortexed 3 minutes and microfuged for 10 sec. Thesupernatant was removed and the microcarriers washed 2 times with 250 μlof 100% EtOH (HPLC grade) by vortexing briefly, centrifuging andremoving the supernatant. The microcarriers are resuspended in 65 μl100% EtOH.

Callus Formation

Embryos were transferred to callus initiation medium with 3 mg/l PPT 1day after bombardment. Embryos were scored for callus initiation at 2and 3 weeks after bombardment. Any responses were transferred to callusmaintenance medium, 2DG4+0.5 2,4-D medium with 3 mg/L PPT. Callusmaintenance medium is N6 major salts, B5 minor salts, MS iron, 2%sucrose, with 0.5 mg/l 2,4-D, 20 mg/l glucose, and 10 ml G4 additionsadded after utoclaving. Embryogenic callus was subcultured every 2 weeksto fresh maintenance medium containing 3 mg/L PPT. All callus wasincubated in the dark at 25° C.

The Type I callus formation response was 18%. Every embryo whichproduced callus was cultured as an individual event giving rise to anindividual line.

Regeneration

After 12 weeks on selection, the tissue was removed from callusmaintenance medium with PPT and was placed on regeneration medium andincubated at 250° C. using a 16 hour light (50 μE .m-2. s-1)/8 hour darkphotoperiod. Regeneration medium is 0.25MS3S5BA (0.25 mg/l 2,4 D, 5 mg/lBAP, MS salts, 3% sucrose) for 2 weeks followed by subculture to MS3Smedium for regeneration of plants. After 4 to 10 weeks, plants wereremoved and put into GA 7's. Our line KC64 0-1, which became the #170 BTevent, produced 55 plants. Our line KC64 0-7, which became the #171 BTevent, produced a total of 33 plants.

Assays

Eleven plants, as they became established in the GA7's, were tested bythe chlorophenol red (CR) test for resistance to PPT as per Shillito, etal, above. This assay utilizes a pH sensitive indicator dye to showwhich cells are growing in the presence of PPT. Cells which grow producea pH change in the media and turn the indicator yellow (from red).Plants expressing the resistance gene to PPT are easily seen in thistest. Plants positive by the CR test were assayed by PCR for thepresence of the synthetic BT gene. (Event 170=37 positive/18 negative;#171=25 positive/8 negative).

Plants positive by PCR for the syn-Bt gene were sent to the phytotron.Once established in the phytotron, they were characterized using insectbioassays and ELISA analysis. Plants were insect bioassayed using astandard European corn borer assay (see below) in which small pieces ofleaf of clipped from a lant and placed in a small petri dish with anumber of ECB neonate larvae. Plants are typically assayed at a heightof about 6 inches. Plants showing 100% mortality to ECB in this assayare characterized further. ELISA data are shown below. Positive plantsare moved to the greenhouse.

Basta screening

Eight of the mature plants from the #170 event were selected forevaluation of Basta (Hoechst) resistance. On one middle leaf per plant,an area approximately 10-14 cm long×the leaf width was painted with 0,0.4, 1.0 or 2.0% (10 ml of 200 g/L diluted to 100 ml with deionizedwater) aqueous Basta containing 2 drops of Tween 20/100 ml. Two plantswere tested per level. Eight wild-type 6N615 plants of the sameapproximate age were treated as controls. All plants were observed at 4and 7 days. All of the control plants eventually died. Throughout thestudy, none of the #170 plants displayed any damage due to theherbicide.

Pollination

All tassel ears, first ear and, if available, the second ear on the #170and #171 plants were pollinated with wild-type 6N615 pollen. At least90% of the plants were female fertile.

Pollen from #171 plants was outcrossed to genotypes 6N615, 5N984, 5NA89,6F010, 5NA56, 2N217AF, 2NDO 1 and 3N961. At least 90% of the plants wereshown to be male fertile.

Embryo Rescue

Embryos from the #171 event have been "rescued." Fourteen to 16 daysafter pollination, the ear tip with 25-50 kernels was cut from the earwith a coping saw. Prior to cutting, the husks were gently peeled awayto expose the upper portion of the ear. The cut end of the ear on theplant was painted with Captan fungicide and the husks replaced. The seedremaining on the plant was allowed to dry naturally.

The excised ear piece was surface sterilized with 20% Clorox for 20minutes and rinsed 3 times with sterile water. Individual embryos wereexcised and plated scutellum side up on B5 medium (Gamborg) containing2% sucrose. B5 vitamins are added to the medium after autoclaving. Fourembryos were plated per GA7 container and the containers incubated inthe dark. When germination occurred, the containers were moved to alight culture room and incubated at 25° C. using a 16 hour light (50 μE.m-2. s-1)/8 hour dark photoperiod. The germination frequency is 94%.

Progeny from 15 plants of the #171 event and 2 of the #176 event wererescued using standard embryo rescue techniques and evaluated. Allplants were evaluated by insect assay. Plants from the #171 event werealso tested in the histochemical GUS assay. In both the insect assay andthe GUS assay, the ratio of segregation of the transgenes was 1:1, asexpected for a single locus insertion event.

Example 48

Analysis of Transgenic Maize Plants

ELISA ASSAY

Detection of cryIA(b) gene expression in transgenic maize is monitoredusing European corn borer(ECB) insect bioassays and ELISA analysis for aquantitative determination of the level of cryIA(b) protein obtained.Quantitative determination of cryIA(b) IP in the leaves of transgenicplants was performed using enzyme-linked immunosorbant assays (ELISA) asdisclosed in Clark M F, Lister R M, Bar-Joseph M: ELISA Techniques. In:Weissbach A, Weissbach H (eds) Methods in Enzymology 118:742-766,Academic Press, Florida (1986). Immunoaffinity purified polyclonalrabbit and goat antibodies specific for the B. thuringiensis subsp.kurstaki IP were used to determine ng IP per mg soluble protein fromcrude extracts of leaf samples. The sensitivity of the double sandwichELISA is 1-5 ng IP per mg soluble protein using 50 ug of total proteinper ELISA microtiter dish well.

Corn extracts were made by grinding leaf tissue in gauze lined plasticbags using a hand held ball-bearing homogenizer (AGDIA, Elkart Ind.) inthe presence of extraction buffer (50 mM Na₂ CO₃ pH 9.5, 100 mM NaCl,0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 μM leupeptin). Proteindetermination was performed using the Bio-Rad (Richmond, Calif.) proteinassay.

Using the above procedure, the primary maize transformants describedabove were analyzed for the presence of cryIA(b) protein using ELISA.These plants varied in height from 6 inches to about three feet at thetime of analysis.

    ______________________________________                                        Plant       Bt ng/ml soluble protein                                                                     5/27/91                                            ______________________________________                                        176-8       0              0                                                  176-10      700            1585                                               176-11      760            2195                                               171-4A      59                                                                171-6       50                                                                171-8       60                                                                171-9       280                                                               171-13      77                                                                171-14A     43                                                                171-14B     60                                                                171-15      55                                                                171-16A     13                                                                171-16B     19                                                                171-18      19                                                                176-30      1160                                                              171-32      980                                                               171-31      166                                                               171-30      370                                                               71-14                                                                         #10 leaf    26                                                                1 leaf      17                                                                plant       171-16                                                            #leaf       40                                                                #1 leaf     120                                                               ______________________________________                                    

EUROPEAN CORN BORER ASSAY

1. One to four 4 cm sections are cut from an extended leaf of a cornplant.

2. Each leaf piece is placed on a moistened filter disc in a 50×9 mmpetri dish.

3. Five neonate European corn borer larvae are placed on each leafpiece. (Making a total of 5-20 larvae per plant.)

4. The petri dishes are incubated at 29.5° C.

5. Leaf feeding damage and mortality data are scored at 24, 48, and 72hours.

Example 49

Expression of Bt Endotoxin in Progeny of Transformed Maize Plants

The transformed maize plants were fully fertile and were crossed withseveral genotypes of maize. Progeny from these crosses were analyzed fortheir ability to kill European corn borer (ECB) in a standard ECBbioassay (described immediately above) as well as for the presence ofthe cryIA(b) protein using ELISA as described above. The ability to killECB and the production of cryIA(b) protein correlated. These traitssegregated to the progeny with a 1:1 ratio, indicating a single site ofinsertion for the active copy of the synthetic gene. This 1:1 ratio wastrue for both the constitutive promoter/synthetic-cryIA(b) plants andthe tissue specific promoter/synthetic-cryIA(b) plants (data not shown).

FIG. 23A is a table containing a small subset of the total number ofprogeny analyzed. This table is representative of a number of differentcrosses.

Insect assays were done with Diatrea saccharalis and Ostrinia nubilalisusing leaf material (as described above) of transgenic progenycontaining a maize optimized CryIA(b) gene. The results of these assaysare shown in FIG. 23B. They demonstrate that the maize optimizedCryIA(b) gene functions in transformed maize to provide resistance toSugarcane borer and Ostrinia nubilalis.

Example 50

EXPRESSION OF THE CRYIA(b) GENE IN MAIZE POLLEN

Progeny of the transformed maize plants containing the chimeric pollenpromoter/synthetic cryIA(b) gene derived from pCIB4431 were grown in thefield to maturity. Pollen was collected and analyzed for the presence ofthe cryIA(b) protein using standard ELISA techniquesd as describedelsewhere. High levels of cryIA(b) protein were detected in the pollen.Progeny from the 35S promoter/synthetic cryIA(b) transformed plant weregrown in the greenhouse. Pollen from these plants was analyzed usingELISA, and cryIA(b) protein was detected. Results are shown below inFIG. 23C.

It is recognized that factors including selection of plant lines, plantgenotypes, synthetic sequences and the like, may also affect expression.

Example 51

EXPRESSION OF THE CRYIA(b) GENE FUSED TO A PITH-PREFERRED PROMOTER.

pCIB4433 (FIG. 36) is a plasmid containing the maize optimized CryIA(b)gene fused with the pith-preferred promoter isolated from maize. Thisplasmid was constructed using a three-way ligation consisting of:

1) pCIB4418, cut with BstEH and BamHI; 1.8 Kb fragment

2) pBtin1, cut with NsiI and BstEII; 5.9 Kb fragment; pBtinl isdescribed elsewhere in this application

3) PCR fragment VI-151 was generated in a PCR reaction using standardconditions as described elsewhere in this application.

PCR primers utilized were:

KE150A28: 5'-ATT CGC ATG CAT GTT TCA TTA TC-3' (SEQ ID NO: 93)

KE151A28: 5'-GCT GGT ACC ACG GAT CCG TCG CTT CTG TGC AAC AAC C-3' (SEQID NO: 94)

After the PCR reaction, the DNA was checked on an agarose gel to makesure the reaction had proceeded properly. DNA was recovered from the PCRreaction using standard conditions described elsewhere and subsequentlycut with the restriction enzymes NsiI and BamHI using standardcondition. After cutting, the fragment was run on a 2% NuSieve gel andthe desired band recovered as described elsewhere. The DNA was used inthe ligation described above.

After ligation (under standard condition), the DNA was transformed intocompetent E. coli cell.

Transformation was carried out using microprojectile bombardmentessentially as described elsewhere in this application. Embryos weretransferred to medium containing 10·μg/ml PPT 24 hours aftermicroprojectile bombardment. Resulting callus was transferred to mediumcontaining 40 μg/ml PPT after four weeks. Plants were regeneratedwithout selection.

A small sample of plants (3-5) was assayed by PCR for each event.Further codes were added to indicate different positions and distancesof embryos with respect to the microprojectile bombardment device.Plants were sent to the greenhouse having the following codes:

    ______________________________________                                        JS21A TOP    Plants B.t. PCR Positive                                         JS21A MID    Plants B.t. PCR Positive                                         JS21C BOT    Plants B.t. PCR Positive                                         JS22D MID    Plants B.t. PCR Positive                                         JS23B MID    Plants B.t. PCR Negative (for control)                           ______________________________________                                    

Leaf samples from the regenerated plants were bioassayed forinsecticidal activity against European corn borer as described inExample 48 with the results shown in FIG. 23D.

ELISA analysis of leaf samples to quantify the level of CryIA(b) proteinexpressed in the leaves was carried out as described in Example 48 withthe results shown in FIG. 23E.

DEPOSITS

The following plasmids have been deposited with the AgriculturalResearch Culture Collection (NRRL)(1818 N. University St., Peoria, Ill.61604) under the provisions of the Budapest Treaty: pCIB4418, pCIB4420,pCIB4429, pCIB4431, pCIB4433, pCIB5601, pCIB3166 and pCIB3171.

The present invention has been described with reference to specificembodiments thereof; however it will be appreciated that numerousvariations, modifications, and embodiments are possible. Accordingly,all such variations, modifications and embodiments are to be regarded asbeing within the spirit and scope of the present invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 94                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3468 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                   #thuringiensis kurstaki Bacillus                                                        (B) STRAIN: HD-1                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..3468                                               #/product= "Full-length nativeN:                                                             cryIA(b)"                                                      #"Appears in Figures 1 and 4 as BTHKURHD."                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGGATAACA ATCCGAACAT CAATGAATGC ATTCCTTATA ATTGTTTAAG TA - #ACCCTGAA         60                                                                          - GTAGAAGTAT TAGGTGGAGA AAGAATAGAA ACTGGTTACA CCCCAATCGA TA - #TTTCCTTG        120                                                                          - TCGCTAACGC AATTTCTTTT GAGTGAATTT GTTCCCGGTG CTGGATTTGT GT - #TAGGACTA        180                                                                          - GTTGATATAA TATGGGGAAT TTTTGGTCCC TCTCAATGGG ACGCATTTCT TG - #TACAAATT        240                                                                          - GAACAGTTAA TTAACCAAAG AATAGAAGAA TTCGCTAGGA ACCAAGCCAT TT - #CTAGATTA        300                                                                          - GAAGGACTAA GCAATCTTTA TCAAATTTAC GCAGAATCTT TTAGAGAGTG GG - #AAGCAGAT        360                                                                          - CCTACTAATC CAGCATTAAG AGAAGAGATG CGTATTCAAT TCAATGACAT GA - #ACAGTGCC        420                                                                          - CTTACAACCG CTATTCCTCT TTTTGCAGTT CAAAATTATC AAGTTCCTCT TT - #TATCAGTA        480                                                                          - TATGTTCAAG CTGCAAATTT ACATTTATCA GTTTTGAGAG ATGTTTCAGT GT - #TTGGACAA        540                                                                          - AGGTGGGGAT TTGATGCCGC GACTATCAAT AGTCGTTATA ATGATTTAAC TA - #GGCTTATT        600                                                                          - GGCAACTATA CAGATCATGC TGTACGCTGG TACAATACGG GATTAGAGCG TG - #TATGGGGA        660                                                                          - CCGGATTCTA GAGATTGGAT AAGATATAAT CAATTTAGAA GAGAATTAAC AC - #TAACTGTA        720                                                                          - TTAGATATCG TTTCTCTATT TCCGAACTAT GATAGTAGAA CGTATCCAAT TC - #GAACAGTT        780                                                                          - TCCCAATTAA CAAGAGAAAT TTATACAAAC CCAGTATTAG AAAATTTTGA TG - #GTAGTTTT        840                                                                          - CGAGGCTCGG CTCAGGGCAT AGAAGGAAGT ATTAGGAGTC CACATTTGAT GG - #ATATACTT        900                                                                          - AACAGTATAA CCATCTATAC GGATGCTCAT AGAGGAGAAT ATTATTGGTC AG - #GGCATCAA        960                                                                          - ATAATGGCTT CTCCTGTAGG GTTTTCGGGG CCAGAATTCA CTTTTCCGCT AT - #ATGGAACT       1020                                                                          - ATGGGAAATG CAGCTCCACA ACAACGTATT GTTGCTCAAC TAGGTCAGGG CG - #TGTATAGA       1080                                                                          - ACATTATCGT CCACTTTATA TAGAAGACCT TTTAATATAG GGATAAATAA TC - #AACAACTA       1140                                                                          - TCTGTTCTTG ACGGGACAGA ATTTGCTTAT GGAACCTCCT CAAATTTGCC AT - #CCGCTGTA       1200                                                                          - TACAGAAAAA GCGGAACGGT AGATTCGCTG GATGAAATAC CGCCACAGAA TA - #ACAACGTG       1260                                                                          - CCACCTAGGC AAGGATTTAG TCATCGATTA AGCCATGTTT CAATGTTTCG TT - #CAGGCTTT       1320                                                                          - AGTAATAGTA GTGTAAGTAT AATAAGAGCT CCTATGTTCT CTTGGATACA TC - #GTAGTGCT       1380                                                                          - GAATTTAATA ATATAATTCC TTCATCACAA ATTACACAAA TACCTTTAAC AA - #AATCTACT       1440                                                                          - AATCTTGGCT CTGGAACTTC TGTCGTTAAA GGACCAGGAT TTACAGGAGG AG - #ATATTCTT       1500                                                                          - CGAAGAACTT CACCTGGCCA GATTTCAACC TTAAGAGTAA ATATTACTGC AC - #CATTATCA       1560                                                                          - CAAAGATATC GGGTAAGAAT TCGCTACGCT TCTACCACAA ATTTACAATT CC - #ATACATCA       1620                                                                          - ATTGACGGAA GACCTATTAA TCAGGGGAAT TTTTCAGCAA CTATGAGTAG TG - #GGAGTAAT       1680                                                                          - TTACAGTCCG GAAGCTTTAG GACTGTAGGT TTTACTACTC CGTTTAACTT TT - #CAAATGGA       1740                                                                          - TCAAGTGTAT TTACGTTAAG TGCTCATGTC TTCAATTCAG GCAATGAAGT TT - #ATATAGAT       1800                                                                          - CGAATTGAAT TTGTTCCGGC AGAAGTAACC TTTGAGGCAG AATATGATTT AG - #AAAGAGCA       1860                                                                          - CAAAAGGCGG TGAATGAGCT GTTTACTTCT TCCAATCAAA TCGGGTTAAA AA - #CAGATGTG       1920                                                                          - ACGGATTATC ATATTGATCA AGTATCCAAT TTAGTTGAGT GTTTATCTGA TG - #AATTTTGT       1980                                                                          - CTGGATGAAA AAAAAGAATT GTCCGAGAAA GTCAAACATG CGAAGCGACT TA - #GTGATGAG       2040                                                                          - CGGAATTTAC TTCAAGATCC AAACTTTAGA GGGATCAATA GACAACTAGA CC - #GTGGCTGG       2100                                                                          - AGAGGAAGTA CGGATATTAC CATCCAAGGA GGCGATGACG TATTCAAAGA GA - #ATTACGTT       2160                                                                          - ACGCTATTGG GTACCTTTGA TGAGTGCTAT CCAACGTATT TATATCAAAA AA - #TAGATGAG       2220                                                                          - TCGAAATTAA AAGCCTATAC CCGTTACCAA TTAAGAGGGT ATATCGAAGA TA - #GTCAAGAC       2280                                                                          - TTAGAAATCT ATTTAATTCG CTACAATGCC AAACACGAAA CAGTAAATGT GC - #CAGGTACG       2340                                                                          - GGTTCCTTAT GGCCGCTTTC AGCCCCAAGT CCAATCGGAA AATGTGCCCA TC - #ATTCCCAT       2400                                                                          - CATTTCTCCT TGGACATTGA TGTTGGATGT ACAGACTTAA ATGAGGACTT AG - #GTGTATGG       2460                                                                          - GTGATATTCA AGATTAAGAC GCAAGATGGC CATGCAAGAC TAGGAAATCT AG - #AATTTCTC       2520                                                                          - GAAGAGAAAC CATTAGTAGG AGAAGCACTA GCTCGTGTGA AAAGAGCGGA GA - #AAAAATGG       2580                                                                          - AGAGACAAAC GTGAAAAATT GGAATGGGAA ACAAATATTG TTTATAAAGA GG - #CAAAAGAA       2640                                                                          - TCTGTAGATG CTTTATTTGT AAACTCTCAA TATGATAGAT TACAAGCGGA TA - #CCAACATC       2700                                                                          - GCGATGATTC ATGCGGCAGA TAAACGCGTT CATAGCATTC GAGAAGCTTA TC - #TGCCTGAG       2760                                                                          - CTGTCTGTGA TTCCGGGTGT CAATGCGGCT ATTTTTGAAG AATTAGAAGG GC - #GTATTTTC       2820                                                                          - ACTGCATTCT CCCTATATGA TGCGAGAAAT GTCATTAAAA ATGGTGATTT TA - #ATAATGGC       2880                                                                          - TTATCCTGCT GGAACGTGAA AGGGCATGTA GATGTAGAAG AACAAAACAA CC - #ACCGTTCG       2940                                                                          - GTCCTTGTTG TTCCGGAATG GGAAGCAGAA GTGTCACAAG AAGTTCGTGT CT - #GTCCGGGT       3000                                                                          - CGTGGCTATA TCCTTCGTGT CACAGCGTAC AAGGAGGGAT ATGGAGAAGG TT - #GCGTAACC       3060                                                                          - ATTCATGAGA TCGAGAACAA TACAGACGAA CTGAAGTTTA GCAACTGTGT AG - #AAGAGGAA       3120                                                                          - GTATATCCAA ACAACACGGT AACGTGTAAT GATTATACTG CGACTCAAGA AG - #AATATGAG       3180                                                                          - GGTACGTACA CTTCTCGTAA TCGAGGATAT GACGGAGCCT ATGAAAGCAA TT - #CTTCTGTA       3240                                                                          - CCAGCTGATT ATGCATCAGC CTATGAAGAA AAAGCATATA CAGATGGACG AA - #GAGACAAT       3300                                                                          - CCTTGTGAAT CTAACAGAGG ATATGGGGAT TACACACCAC TACCAGCTGG CT - #ATGTGACA       3360                                                                          - AAAGAATTAG AGTACTTCCC AGAAACCGAT AAGGTATGGA TTGAGATCGG AG - #AAACGGAA       3420                                                                          #              3468ACAG CGTGGAATTA CTTCTTATGG AGGAATAA                        - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3468 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..3468                                               #/product= "Full-length pure maize                                            #synthetic Bt" optimized                                                      #"Disclosed in Figure 3 as syn1T.mze"                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CA - #ACCCCGAG         60                                                                          - GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CA - #TCAGCCTG        120                                                                          - AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GC - #TGGGCCTG        180                                                                          - GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GG - #TGCAGATC        240                                                                          - GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CA - #GCCGCCTG        300                                                                          - GAGGGCCTGA GCAACCTGTA CCAGATCTAC GCCGAGAGCT TCCGCGAGTG GG - #AGGCCGAC        360                                                                          - CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GA - #ACAGCGCC        420                                                                          - CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GC - #TGAGCGTG        480                                                                          - TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTGAGCGT GT - #TCGGCCAG        540                                                                          - CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CC - #GCCTGATC        600                                                                          - GGCAACTACA CCGACCACGC CGTGCGCTGG TACAACACCG GCCTGGAGCG CG - #TGTGGGGC        660                                                                          - CCCGACAGCC GCGACTGGAT CCGCTACAAC CAGTTCCGCC GCGAGCTGAC CC - #TGACCGTG        720                                                                          - CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CC - #GCACCGTG        780                                                                          - AGCCAGCTGA CCCGCGAGAT CTACACCAAC CCCGTGCTGG AGAACTTCGA CG - #GCAGCTTC        840                                                                          - CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GG - #ACATCCTG        900                                                                          - AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CG - #GCCACCAG        960                                                                          - ATCATGGCCA GCCCCGTGGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GT - #ACGGCACC       1020                                                                          - ATGGGCAACG CCGCCCCCCA GCAGCGCATC GTGGCCCAGC TGGGCCAGGG CG - #TGTACCGC       1080                                                                          - ACCCTGAGCA GCACCCTGTA CCGCCGCCCC TTCAACATCG GCATCAACAA CC - #AGCAGCTG       1140                                                                          - AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CA - #GCGCCGTG       1200                                                                          - TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCCCAGAA CA - #ACAACGTG       1260                                                                          - CCCCCCCGCC AGGGCTTCAG CCACCGCCTG AGCCACGTGA GCATGTTCCG CA - #GCGGCTTC       1320                                                                          - AGCAACAGCA GCGTGAGCAT CATCCGCGCC CCCATGTTCA GCTGGATCCA CC - #GCAGCGCC       1380                                                                          - GAGTTCAACA ACATCATCCC CAGCAGCCAG ATCACCCAGA TCCCCCTGAC CA - #AGAGCACC       1440                                                                          - AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CG - #ACATCCTG       1500                                                                          - CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CC - #CCCTGAGC       1560                                                                          - CAGCGCTACC GCGTGCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CC - #ACACCAGC       1620                                                                          - ATCGACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CG - #GCAGCAAC       1680                                                                          - CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CA - #GCAACGGC       1740                                                                          - AGCAGCGTGT TCACCCTGAG CGCCCACGTG TTCAACAGCG GCAACGAGGT GT - #ACATCGAC       1800                                                                          - CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GG - #AGCGCGCC       1860                                                                          - CAGAAGGCCG TGAACGAGCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA GA - #CCGACGTG       1920                                                                          - ACCGACTACC ACATCGACCA GGTGAGCAAC CTGGTGGAGT GCCTGAGCGA CG - #AGTTCTGC       1980                                                                          - CTGGACGAGA AGAAGGAGCT GAGCGAGAAG GTGAAGCACG CCAAGCGCCT GA - #GCGACGAG       2040                                                                          - CGCAACCTGC TGCAGGACCC CAACTTCCGC GGCATCAACC GCCAGCTGGA CC - #GCGGCTGG       2100                                                                          - CGCGGCAGCA CCGACATCAC CATCCAGGGC GGCGACGACG TGTTCAAGGA GA - #ACTACGTG       2160                                                                          - ACCCTGCTGG GCACCTTCGA CGAGTGCTAC CCCACCTACC TGTACCAGAA GA - #TCGACGAG       2220                                                                          - AGCAAGCTGA AGGCCTACAC CCGCTACCAG CTGCGCGGCT ACATCGAGGA CA - #GCCAGGAC       2280                                                                          - CTGGAGATCT ACCTGATCCG CTACAACGCC AAGCACGAGA CCGTGAACGT GC - #CCGGCACC       2340                                                                          - GGCAGCCTGT GGCCCCTGAG CGCCCCCAGC CCCATCGGCA AGTGCGCCCA CC - #ACAGCCAC       2400                                                                          - CACTTCAGCC TGGACATCGA CGTGGGCTGC ACCGACCTGA ACGAGGACCT GG - #GCGTGTGG       2460                                                                          - GTGATCTTCA AGATCAAGAC CCAGGACGGC CACGCCCGCC TGGGCAACCT GG - #AGTTCCTG       2520                                                                          - GAGGAGAAGC CCCTGGTGGG CGAGGCCCTG GCCCGCGTGA AGCGCGCCGA GA - #AGAAGTGG       2580                                                                          - CGCGACAAGC GCGAGAAGCT GGAGTGGGAG ACCAACATCG TGTACAAGGA GG - #CCAAGGAG       2640                                                                          - AGCGTGGACG CCCTGTTCGT GAACAGCCAG TACGACCGCC TGCAGGCCGA CA - #CCAACATC       2700                                                                          - GCCATGATCC ACGCCGCCGA CAAGCGCGTG CACAGCATCC GCGAGGCCTA CC - #TGCCCGAG       2760                                                                          - CTGAGCGTGA TCCCCGGCGT GAACGCCGCC ATCTTCGAGG AGCTGGAGGG CC - #GCATCTTC       2820                                                                          - ACCGCCTTCA GCCTGTACGA CGCCCGCAAC GTGATCAAGA ACGGCGACTT CA - #ACAACGGC       2880                                                                          - CTGAGCTGCT GGAACGTGAA GGGCCACGTG GACGTGGAGG AGCAGAACAA CC - #ACCGCAGC       2940                                                                          - GTGCTGGTGG TGCCCGAGTG GGAGGCCGAG GTGAGCCAGG AGGTGCGCGT GT - #GCCCCGGC       3000                                                                          - CGCGGCTACA TCCTGCGCGT GACCGCCTAC AAGGAGGGCT ACGGCGAGGG CT - #GCGTGACC       3060                                                                          - ATCCACGAGA TCGAGAACAA CACCGACGAG CTGAAGTTCA GCAACTGCGT GG - #AGGAGGAG       3120                                                                          - GTGTACCCCA ACAACACCGT GACCTGCAAC GACTACACCG CCACCCAGGA GG - #AGTACGAG       3180                                                                          - GGCACCTACA CCAGCCGCAA CCGCGGCTAC GACGGCGCCT ACGAGAGCAA CA - #GCAGCGTG       3240                                                                          - CCCGCCGACT ACGCCAGCGC CTACGAGGAG AAGGCCTACA CCGACGGCCG CC - #GCGACAAC       3300                                                                          - CCCTGCGAGA GCAACCGCGG CTACGGCGAC TACACCCCCC TGCCCGCCGG CT - #ACGTGACC       3360                                                                          - AAGGAGCTGG AGTACTTCCC CGAGACCGAC AAGGTGTGGA TCGAGATCGG CG - #AGACCGAG       3420                                                                          #              3468ACAG CGTGGAGCTG CTGCTGATGG AGGAGTAG                        - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1947 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..1947                                               #/product= "Truncated synthetic:                                                             maize opt - #imized cryIA(b) gene"                             #"Disclosed in Figures 1, 2, 3, 4 and 5 as - # bssyn."                        -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CA - #ACCCCGAG         60                                                                          - GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CA - #TCAGCCTG        120                                                                          - AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GC - #TGGGCCTG        180                                                                          - GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GG - #TGCAGATC        240                                                                          - GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CA - #GCCGCCTG        300                                                                          - GAGGGCCTGA GCAACCTGTA CCAAATCTAC GCCGAGAGCT TCCGCGAGTG GG - #AGGCCGAC        360                                                                          - CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GA - #ACAGCGCC        420                                                                          - CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GC - #TGAGCGTG        480                                                                          - TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTCAGCGT GT - #TCGGCCAG        540                                                                          - CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CC - #GCCTGATC        600                                                                          - GGCAACTACA CCGACCACGC CGTGCGCTGG TACAACACCG GCCTGGAGCG CG - #TGTGGGGT        660                                                                          - CCCGACAGCC GCGACTGGAT CAGGTACAAC CAGTTCCGCC GCGAGCTGAC CC - #TGACCGTG        720                                                                          - CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CC - #GCACCGTG        780                                                                          - AGCCAGCTGA CCCGCGAGAT TTACACCAAC CCCGTGCTGG AGAACTTCGA CG - #GCAGCTTC        840                                                                          - CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GG - #ACATCCTG        900                                                                          - AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CG - #GCCACCAG        960                                                                          - ATCATGGCCA GCCCCGTCGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GT - #ACGGCACC       1020                                                                          - ATGGGCAACG CTGCACCTCA GCAGCGCATC GTGGCACAGC TGGGCCAGGG AG - #TGTACCGC       1080                                                                          - ACCCTGAGCA GCACCCTGTA CCGTCGACCT TTCAACATCG GCATCAACAA CC - #AGCAGCTG       1140                                                                          - AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CA - #GCGCCGTG       1200                                                                          - TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCTCAGAA CA - #ACAACGTG       1260                                                                          - CCACCTCGAC AGGGCTTCAG CCACCGTCTG AGCCACGTGA GCATGTTCCG CA - #GTGGCTTC       1320                                                                          - AGCAACAGCA GCGTGAGCAT CATCCGTGCA CCTATGTTCA GCTGGATTCA CC - #GCAGTGCC       1380                                                                          - GAGTTCAACA ACATCATCCC CAGCAGCCAA ATCACCCAGA TCCCCCTGAC CA - #AGAGCACC       1440                                                                          - AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CG - #ACATCCTG       1500                                                                          - CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CC - #CCCTGAGC       1560                                                                          - CAGCGCTACC GCGTCCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CC - #ACACCAGC       1620                                                                          - ATCGACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CG - #GCAGCAAC       1680                                                                          - CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CA - #GCAACGGC       1740                                                                          - AGCAGCGTGT TCACCCTGAG CGCCCACGTG TTCAACAGCG GCAACGAGGT GT - #ACATCGAC       1800                                                                          - CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GG - #AGAGGGCT       1860                                                                          - CAGAAGGCCG TGAACGAGCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA GA - #CCGACGTG       1920                                                                          #           1947   ATCA GGTGTAG                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3468 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..3468                                               #/product= "Full length synthetic                                                            maize opt - #imized"                                           #"Disclosed in Figure 3 as synful.mod.  This                                  #is identical to flsynbt.fin as disclosed in                                                 Figure 1. - #"                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CA - #ACCCCGAG         60                                                                          - GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CA - #TCAGCCTG        120                                                                          - AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GC - #TGGGCCTG        180                                                                          - GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GG - #TGCAGATC        240                                                                          - GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CA - #GCCGCCTG        300                                                                          - GAGGGCCTGA GCAACCTGTA CCAAATCTAC GCCGAGAGCT TCCGCGAGTG GG - #AGGCCGAC        360                                                                          - CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GA - #ACAGCGCC        420                                                                          - CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GC - #TGAGCGTG        480                                                                          - TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTCAGCGT GT - #TCGGCCAG        540                                                                          - CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CC - #GCCTGATC        600                                                                          - GGCAACTACA CCGACCACGC CGTGCGCTGG TACAACACCG GCCTGGAGCG CG - #TGTGGGGT        660                                                                          - CCCGACAGCC GCGACTGGAT CAGGTACAAC CAGTTCCGCC GCGAGCTGAC CC - #TGACCGTG        720                                                                          - CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CC - #GCACCGTG        780                                                                          - AGCCAGCTGA CCCGCGAGAT TTACACCAAC CCCGTGCTGG AGAACTTCGA CG - #GCAGCTTC        840                                                                          - CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GG - #ACATCCTG        900                                                                          - AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CG - #GCCACCAG        960                                                                          - ATCATGGCCA GCCCCGTCGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GT - #ACGGCACC       1020                                                                          - ATGGGCAACG CTGCACCTCA GCAGCGCATC GTGGCACAGC TGGGCCAGGG AG - #TGTACCGC       1080                                                                          - ACCCTGAGCA GCACCCTGTA CCGTCGACCT TTCAACATCG GCATCAACAA CC - #AGCAGCTG       1140                                                                          - AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CA - #GCGCCGTG       1200                                                                          - TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCTCAGAA CA - #ACAACGTG       1260                                                                          - CCACCTCGAC AGGGCTTCAG CCACCGTCTG AGCCACGTGA GCATGTTCCG CA - #GTGGCTTC       1320                                                                          - AGCAACAGCA GCGTGAGCAT CATCCGTGCA CCTATGTTCA GCTGGATTCA CC - #GCAGTGCC       1380                                                                          - GAGTTCAACA ACATCATCCC CAGCAGCCAG ATCACCCAGA TCCCCCTGAC CA - #AGAGCACC       1440                                                                          - AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CG - #ACATCCTG       1500                                                                          - CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CC - #CCCTGAGC       1560                                                                          - CAGCGCTACC GCGTCCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CC - #ACACCAGC       1620                                                                          - ATCGACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CG - #GCAGCAAC       1680                                                                          - CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CA - #GCAACGGC       1740                                                                          - AGCAGCGTGT TCACCCTGAG CGCCCACGTG TTCAACAGCG GCAACGAGGT GT - #ACATCGAC       1800                                                                          - CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GG - #AGAGGGCT       1860                                                                          - CAGAAGGCCG TGAACGAGCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA GA - #CCGACGTG       1920                                                                          - ACCGACTACC ACATCGATCA GGTGAGCAAC CTGGTGGAGT GCCTGAGCGA CG - #AGTTCTGC       1980                                                                          - CTGGACGAGA AGAAGGAGCT GAGCGAGAAG GTGAAGCACG CCAAGCGCCT GA - #GCGACGAG       2040                                                                          - CGCAACCTGC TGCAGGACCC CAACTTCCGC GGCATCAACC GCCAGCTGGA CC - #GCGGCTGG       2100                                                                          - CGCGGCAGCA CCGACATCAC CATCCAGGGC GGCGACGACG TGTTCAAGGA GA - #ACTACGTG       2160                                                                          - ACCCTGCTGG GCACCTTCGA CGAGTGCTAC CCCACCTACC TGTACCAGAA GA - #TCGACGAG       2220                                                                          - AGCAAGCTGA AGGCCTACAC CCGCTACCAG CTGCGCGGCT ACATCGAGGA CA - #GCCAGGAC       2280                                                                          - CTGGAGATCT ACCTGATCCG CTACAACGCC AAGCACGAGA CCGTGAACGT GC - #CCGGCACC       2340                                                                          - GGCAGCCTGT GGCCCCTGAG CGCCCCCAGC CCCATCGGCA AGTGCGCCCA CC - #ACAGCCAC       2400                                                                          - CACTTCAGCC TGGACATCGA CGTGGGCTGC ACCGACCTGA ACGAGGACCT GG - #GCGTGTGG       2460                                                                          - GTGATCTTCA AGATCAAGAC CCAGGACGGC CACGCCCGCC TGGGCAACCT GG - #AGTTCCTG       2520                                                                          - GAGGAGAAGC CCCTGGTGGG CGAGGCCCTG GCCCGCGTGA AGCGCGCCGA GA - #AGAAGTGG       2580                                                                          - CGCGACAAGC GCGAGAAGCT GGAGTGGGAG ACCAACATCG TGTACAAGGA GG - #CCAAGGAG       2640                                                                          - AGCGTGGACG CCCTGTTCGT GAACAGCCAG TACGACCGCC TGCAGGCCGA CA - #CCAACATC       2700                                                                          - GCCATGATCC ACGCCGCCGA CAAGCGCGTG CACAGCATTC GCGAGGCCTA CC - #TGCCCGAG       2760                                                                          - CTGAGCGTGA TCCCCGGCGT GAACGCCGCC ATCTTCGAGG AGCTGGAGGG CC - #GCATCTTC       2820                                                                          - ACCGCCTTCA GCCTGTACGA CGCCCGCAAC GTGATCAAGA ACGGCGACTT CA - #ACAACGGC       2880                                                                          - CTGAGCTGCT GGAACGTGAA GGGCCACGTG GACGTGGAGG AGCAGAACAA CC - #ACCGCAGC       2940                                                                          - GTGCTGGTGG TGCCCGAGTG GGAGGCCGAG GTGAGCCAGG AGGTGCGCGT GT - #GCCCCGGC       3000                                                                          - CGCGGCTACA TCCTGCGCGT GACCGCCTAC AAGGAGGGCT ACGGCGAGGG CT - #GCGTGACC       3060                                                                          - ATCCACGAGA TCGAGAACAA CACCGACGAG CTCAAGTTCA GCAACTGCGT GG - #AGGAGGAG       3120                                                                          - GTGTACCCCA ACAACACCGT GACCTGCAAC GACTACACCG CCACCCAGGA GG - #AGTACGAG       3180                                                                          - GGCACCTACA CCAGCCGCAA CCGCGGCTAC GACGGCGCCT ACGAGAGCAA CA - #GCAGCGTG       3240                                                                          - CCCGCCGACT ACGCCAGCGC CTACGAGGAG AAGGCCTACA CCGACGGCCG CC - #GCGACAAC       3300                                                                          - CCCTGCGAGA GCAACCGCGG CTACGGCGAC TACACCCCCC TGCCCGCCGG CT - #ACGTGACC       3360                                                                          - AAGGAGCTGG AGTACTTCCC CGAGACCGAC AAGGTGTGGA TCGAGATCGG CG - #AGACCGAG       3420                                                                          #              3468ACAG CGTGGAGCTG CTGCTGATGG AGGAGTAG                        - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1845 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..1845                                               #/note= "This is the synthetic Bt                                                            gene acco - #rding to Perlak et al. as shown in Figures 4      - # and                                                                                      5 as P - #MONBT."                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - ATGGACAACA ACCCAAACAT CAACGAATGC ATTCCATACA ACTGCTTGAG TA - #ACCCAGAA         60                                                                          - GTTGAAGTAC TTGGTGGAGA ACGCATTGAA ACCGGTTACA CTCCCATCGA CA - #TCTCCTTG        120                                                                          - TCCTTGACAC AGTTTCTGCT CAGCGAGTTC GTGCCAGGTG CTGGGTTCGT TC - #TCGGACTA        180                                                                          - GTTGACATCA TCTGGGGTAT CTTTGGTCCA TCTCAATGGG ATGCATTCCT GG - #TGCAAATT        240                                                                          - GAGCAGTTGA TCAACCAGAG GATCGAAGAG TTCGCCAGGA ACCAGGCCAT CT - #CTAGGTTG        300                                                                          - GAAGGATTGA GCAATCTCTA CCAAATCTAT GCAGAGAGCT TCAGAGAGTG GG - #AAGCCGAT        360                                                                          - CCTACTAACC CAGCTCTCCG CGAGGAAATG CGTATTCAAT TCAACGACAT GA - #ACAGCGCC        420                                                                          - TTGACCACAG CTATCCCATT GTTCGCAGTC CAGAACTACC AAGTTCCTCT CT - #TGTCCGTG        480                                                                          - TACGTTCAAG CAGCTAATCT TCACCTCAGC GTGCTTCGAG ACGTTAGCGT GT - #TTGGGCAA        540                                                                          - AGGTGGGGAT TCGATGCTGC AACCATCAAT AGCCGTTACA ACGACCTTAC TA - #GGCTGATT        600                                                                          - GGAAACTACA CCGACCACGC TGTTCGTTGG TACAACACTG GCTTGGAGCG TG - #TCTGGGGT        660                                                                          - CCTGATTCTA GAGATTGGAT TAGATACAAC CAGTTCAGGA GAGAATTGAC CC - #TCACAGTT        720                                                                          - TTGGACATTG TGTCTCTCTT CCCGAACTAT GACTCCAGAA CCTACCCTAT CC - #GTACAGTG        780                                                                          - TCCCAACTTA CCAGAGAAAT CTATACTAAC CCAGTTCTTG AGAACTTCGA CG - #GTAGCTTC        840                                                                          - CGTGGTTCTG CCCAAGGTAT CGAAGGCTCC ATCAGGAGCC CACACTTGAT GG - #ACATCTTG        900                                                                          - AACAGCATAA CTATCTACAG CGATGCTCAC AGAGGAGAGT ATTACTGGTC TG - #GACACCAG        960                                                                          - ATCATGGCCT CTCCAGTTGG ATTCAGCGGG CCCGAGTTTA CCTTTCCTCT CT - #ATGGAACT       1020                                                                          - ATGGGAAACG CCGCTCCACA ACAACGTATC GTTGCTCAAC TAGGTCAGGG TG - #TCTACAGA       1080                                                                          - ACCTTGTCTT CCACCTTGTA CAGAAGACCC TTCAATATCG GTATCAACAA CC - #AGCAACTT       1140                                                                          - TCCGTTCTTG ACGGAACAGA GTTCGCCTAT GGAACCTCTT CTAACTTGCC AT - #CCGCTGTT       1200                                                                          - TACAGAAAGA GCGGAACCGT TGATTCCTTG GACGAAATCC CACCACAGAA CA - #ACAATGTG       1260                                                                          - CCACCCAGGC AAGGATTCTC CCACAGGTTG AGCCACGTGT CCATGTTCCG TT - #CCGGATTC       1320                                                                          - AGCAACAGTT CCGTGAGCAT CATCAGAGCT CCTATGTTCT CATGGATTCA TC - #GTAGTGCT       1380                                                                          - GAGTTCAACA ATATCATTCC TTCCTCTCAA ATCACCCAAA TCCCATTGAC CA - #AGTCTACT       1440                                                                          - AACCTTGGAT CTGGAACTTC TGTCGTGAAA GGACCAGGCT TCACAGGAGG TG - #ATATTCTT       1500                                                                          - AGAAGAACTT CTCCTGGCCA GATTAGCACC CTCAGAGTTA ACATCACTGC AC - #CACTTTCT       1560                                                                          - CAAAGATATC GTGTCAGGAT TCGTTACGCA TCTACCACTA ACTTGCAATT CC - #ACACCTCC       1620                                                                          - ATCGACGGAA GGCCTATCAA TCAGGGTAAC TTCTCCGCAA CCATGTCAAG CG - #GCAGCAAC       1680                                                                          - TTGCAATCCG GCAGCTTCAG AACCGTCGGT TTCACTACTC CTTTCAACTT CT - #CTAACGGA       1740                                                                          - TCAAGCGTTT TCACCCTTAG CGCTCATGTG TTCAATTCTG GCAATGAAGT GT - #ACATTGAC       1800                                                                          #                1845GC CGAAGTTACC TTCGAGGCTG AGTAC                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3624 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3621                                               #/product= "Full-length, maizeN:                                              #cryIB"        optmized                                                       #"Disclosed in Figure 6."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - ATG GAC CTG CTG CCC GAC GCC CGC ATC GAG GA - #C AGC CTG TGC ATC GCC           48                                                                          Met Asp Leu Leu Pro Asp Ala Arg Ile Glu As - #p Ser Leu Cys Ile Ala           #                 15                                                          - GAG GGC AAC AAC ATC GAC CCC TTC GTG AGC GC - #C AGC ACC GTG CAG ACC           96                                                                          Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Al - #a Ser Thr Val Gln Thr           #             30                                                              - GGC ATC AAC ATC GCC GGC CGC ATC CTG GGC GT - #G CTG GGC GTG CCC TTC          144                                                                          Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Va - #l Leu Gly Val Pro Phe           #         45                                                                  - GCC GGC CAG CTG GCC AGC TTC TAC AGC TTC CT - #G GTG GGC GAG CTG TGG          192                                                                          Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Le - #u Val Gly Glu Leu Trp           #     60                                                                      - CCC CGC GGC CGC GAC CAG TGG GAG ATC TTC CT - #G GAG CAC GTG GAG CAG          240                                                                          Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe Le - #u Glu His Val Glu Gln           # 80                                                                          - CTG ATC AAC CAG CAG ATC ACC GAG AAC GCC CG - #C AAC ACC GCC CTG GCC          288                                                                          Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Ar - #g Asn Thr Ala Leu Ala           #                 95                                                          - CGC CTG CAG GGC CTG GGC GAC AGC TTC CGC GC - #C TAC CAG CAG AGC CTG          336                                                                          Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Al - #a Tyr Gln Gln Ser Leu           #           110                                                               - GAG GAC TGG CTG GAG AAC CGC GAC GAC GCC CG - #C ACC CGC AGC GTG CTG          384                                                                          Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Ar - #g Thr Arg Ser Val Leu           #       125                                                                   - TAC ACC CAG TAC ATC GCC CTG GAG CTG GAC TT - #C CTG AAC GCC ATG CCC          432                                                                          Tyr Thr Gln Tyr Ile Ala Leu Glu Leu Asp Ph - #e Leu Asn Ala Met Pro           #   140                                                                       - CTG TTC GCC ATC CGC AAC CAG GAG GTG CCC CT - #G CTG ATG GTG TAC GCC          480                                                                          Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Le - #u Leu Met Val Tyr Ala           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - CAG GCC GCC AAC CTG CAC CTG CTG CTG CTG CG - #C GAC GCC AGC CTG TTC          528                                                                          Gln Ala Ala Asn Leu His Leu Leu Leu Leu Ar - #g Asp Ala Ser Leu Phe           #               175                                                           - GGC AGC GAG TTC GGC CTG ACC AGC CAG GAG AT - #C CAG CGC TAC TAC GAG          576                                                                          Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Il - #e Gln Arg Tyr Tyr Glu           #           190                                                               - CGC CAG GTG GAG CGC ACC CGC GAC TAC AGC GA - #C TAC TGC GTG GAG TGG          624                                                                          Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser As - #p Tyr Cys Val Glu Trp           #       205                                                                   - TAC AAC ACC GGC CTG AAC AGC CTG CGC GGC AC - #C AAC GCC GCC AGC TGG          672                                                                          Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Th - #r Asn Ala Ala Ser Trp           #   220                                                                       - GTG CGC TAC AAC CAG TTC CGC CGC GAC CTG AC - #C CTG GGC GTG CTG GAC          720                                                                          Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Th - #r Leu Gly Val Leu Asp           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - CTG GTG GCC CTG TTC CCC AGC TAC GAC ACC CG - #C ACC TAC CCC ATC AAC          768                                                                          Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Ar - #g Thr Tyr Pro Ile Asn           #               255                                                           - ACC AGC GCC CAG CTG ACC CGC GAG GTG TAC AC - #C GAC GCC ATC GGC GCC          816                                                                          Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Th - #r Asp Ala Ile Gly Ala           #           270                                                               - ACC GGC GTG AAC ATG GCC AGC ATG AAC TGG TA - #C AAC AAC AAC GCC CCC          864                                                                          Thr Gly Val Asn Met Ala Ser Met Asn Trp Ty - #r Asn Asn Asn Ala Pro           #       285                                                                   - AGC TTC AGC GCC ATC GAG GCC GCC GCC ATC CG - #C AGC CCC CAC CTG CTG          912                                                                          Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Ar - #g Ser Pro His Leu Leu           #   300                                                                       - GAC TTC CTG GAG CAG CTG ACC ATC TTC AGC GC - #C AGC AGC CGC TGG AGC          960                                                                          Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Al - #a Ser Ser Arg Trp Ser           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAC ACC CGC CAC ATG ACC TAC TGG CGC GGC CA - #C ACC ATC CAG AGC CGC         1008                                                                          Asn Thr Arg His Met Thr Tyr Trp Arg Gly Hi - #s Thr Ile Gln Ser Arg           #               335                                                           - CCC ATC GGC GGC GGC CTG AAC ACC AGC ACC CA - #C GGC GCC ACC AAC ACC         1056                                                                          Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr Hi - #s Gly Ala Thr Asn Thr           #           350                                                               - AGC ATC AAC CCC GTG ACC CTG CGC TTC GCC AG - #C CGC GAC GTG TAC CGC         1104                                                                          Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Se - #r Arg Asp Val Tyr Arg           #       365                                                                   - ACC GAG AGC TAC GCC GGC GTG CTG CTG TGG GG - #C ATC TAC CTG GAG CCC         1152                                                                          Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gl - #y Ile Tyr Leu Glu Pro           #   380                                                                       - ATC CAC GGC GTG CCC ACC GTG CGC TTC AAC TT - #C ACC AAC CCC CAG AAC         1200                                                                          Ile His Gly Val Pro Thr Val Arg Phe Asn Ph - #e Thr Asn Pro Gln Asn           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - ATC AGC GAC CGC GGC ACC GCC AAC TAC AGC CA - #G CCC TAC GAG AGC CCC         1248                                                                          Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gl - #n Pro Tyr Glu Ser Pro           #               415                                                           - GGC CTG CAG CTG AAG GAC AGC GAG ACC GAG CT - #G CCC CCC GAG ACC ACC         1296                                                                          Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu Le - #u Pro Pro Glu Thr Thr           #           430                                                               - GAG CGC CCC AAC TAC GAG AGC TAC AGC CAC CG - #C CTG AGC CAC ATC GGC         1344                                                                          Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Ar - #g Leu Ser His Ile Gly           #       445                                                                   - ATC ATC CTG CAG AGC CGC GTG AAC GTG CCC GT - #G TAC AGC TGG ACC CAC         1392                                                                          Ile Ile Leu Gln Ser Arg Val Asn Val Pro Va - #l Tyr Ser Trp Thr His           #   460                                                                       - CGC AGC GCC GAC CGC ACC AAC ACC ATC GGC CC - #C AAC CGC ATC ACC CAG         1440                                                                          Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pr - #o Asn Arg Ile Thr Gln           465                 4 - #70                 4 - #75                 4 -       #80                                                                           - ATC CCC ATG GTG AAG GCC AGC GAG CTG CCC CA - #G GGC ACC ACC GTG GTG         1488                                                                          Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gl - #n Gly Thr Thr Val Val           #               495                                                           - CGC GGC CCC GGC TTC ACC GGC GGC GAC ATC CT - #G CGC CGC ACC AAC ACC         1536                                                                          Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Le - #u Arg Arg Thr Asn Thr           #           510                                                               - GGC GGC TTC GGC CCC ATC CGC GTG ACC GTG AA - #C GGC CCC CTG ACC CAG         1584                                                                          Gly Gly Phe Gly Pro Ile Arg Val Thr Val As - #n Gly Pro Leu Thr Gln           #       525                                                                   - CGC TAC CGC ATC GGC TTC CGC TAC GCC AGC AC - #C GTG GAC TTC GAC TTC         1632                                                                          Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Th - #r Val Asp Phe Asp Phe           #   540                                                                       - TTC GTG AGC CGC GGC GGC ACC ACC GTG AAC AA - #C TTC CGC TTC CTG CGC         1680                                                                          Phe Val Ser Arg Gly Gly Thr Thr Val Asn As - #n Phe Arg Phe Leu Arg           545                 5 - #50                 5 - #55                 5 -       #60                                                                           - ACC ATG AAC AGC GGC GAC GAG CTG AAG TAC GG - #C AAC TTC GTG CGC CGC         1728                                                                          Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gl - #y Asn Phe Val Arg Arg           #               575                                                           - GCC TTC ACC ACC CCC TTC ACC TTC ACC CAG AT - #C CAG GAC ATC ATC CGC         1776                                                                          Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Il - #e Gln Asp Ile Ile Arg           #           590                                                               - ACC AGC ATC CAG GGC CTG AGC GGC AAC GGC GA - #G GTG TAC ATC GAC AAG         1824                                                                          Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly Gl - #u Val Tyr Ile Asp Lys           #       605                                                                   - ATC GAG ATC ATC CCC GTG ACC GCC ACC TTC GA - #G GCC GAG TAC GAC CTG         1872                                                                          Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Gl - #u Ala Glu Tyr Asp Leu           #   620                                                                       - GAG CGC GCC CAG GAG GCC GTG AAC GCC CTG TT - #C ACC AAC ACC AAC CCC         1920                                                                          Glu Arg Ala Gln Glu Ala Val Asn Ala Leu Ph - #e Thr Asn Thr Asn Pro           625                 6 - #30                 6 - #35                 6 -       #40                                                                           - CGC CGC CTG AAG ACC GAC GTG ACC GAC TAC CA - #C ATC GAC CAG GTG AGC         1968                                                                          Arg Arg Leu Lys Thr Asp Val Thr Asp Tyr Hi - #s Ile Asp Gln Val Ser           #               655                                                           - AAC CTG GTG GCC TGC CTG AGC GAC GAG TTC TG - #C CTG GAC GAG AAG CGC         2016                                                                          Asn Leu Val Ala Cys Leu Ser Asp Glu Phe Cy - #s Leu Asp Glu Lys Arg           #           670                                                               - GAG CTG CTG GAG AAG GTG AAG TAC GCC AAG CG - #C CTG AGC GAC GAG CGC         2064                                                                          Glu Leu Leu Glu Lys Val Lys Tyr Ala Lys Ar - #g Leu Ser Asp Glu Arg           #       685                                                                   - AAC CTG CTG CAG GAC CCC AAC TTC ACC AGC AT - #C AAC AAG CAG CCC GAC         2112                                                                          Asn Leu Leu Gln Asp Pro Asn Phe Thr Ser Il - #e Asn Lys Gln Pro Asp           #   700                                                                       - TTC ATC AGC ACC AAC GAG CAG AGC AAC TTC AC - #C AGC ATC CAC GAG CAG         2160                                                                          Phe Ile Ser Thr Asn Glu Gln Ser Asn Phe Th - #r Ser Ile His Glu Gln           705                 7 - #10                 7 - #15                 7 -       #20                                                                           - AGC GAG CAC GGC TGG TGG GGC AGC GAG AAC AT - #C ACC ATC CAG GAG GGC         2208                                                                          Ser Glu His Gly Trp Trp Gly Ser Glu Asn Il - #e Thr Ile Gln Glu Gly           #               735                                                           - AAC GAC GTG TTC AAG GAG AAC TAC GTG ACC CT - #G CCC GGC ACC TTC AAC         2256                                                                          Asn Asp Val Phe Lys Glu Asn Tyr Val Thr Le - #u Pro Gly Thr Phe Asn           #           750                                                               - GAG TGC TAC CCC ACC TAC CTG TAC CAG AAG AT - #C GGC GAG AGC GAG CTG         2304                                                                          Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Il - #e Gly Glu Ser Glu Leu           #       765                                                                   - AAG GCC TAC ACC CGC TAC CAG CTG CGC GGC TA - #C ATC GAG GAC AGC CAG         2352                                                                          Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Ty - #r Ile Glu Asp Ser Gln           #   780                                                                       - GAC CTG GAG ATC TAC CTG ATC CGC TAC AAC GC - #C AAG CAC GAG ACC CTG         2400                                                                          Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Al - #a Lys His Glu Thr Leu           785                 7 - #90                 7 - #95                 8 -       #00                                                                           - GAC GTG CCC GGC ACC GAG AGC CTG TGG CCC CT - #G AGC GTG GAG AGC CCC         2448                                                                          Asp Val Pro Gly Thr Glu Ser Leu Trp Pro Le - #u Ser Val Glu Ser Pro           #               815                                                           - ATC GGC CGC TGC GGC GAG CCC AAC CGC TGC GC - #C CCC CAC TTC GAG TGG         2496                                                                          Ile Gly Arg Cys Gly Glu Pro Asn Arg Cys Al - #a Pro His Phe Glu Trp           #           830                                                               - AAC CCC GAC CTG GAC TGC AGC TGC CGC GAC GG - #C GAG AAG TGC GCC CAC         2544                                                                          Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gl - #y Glu Lys Cys Ala His           #       845                                                                   - CAC AGC CAC CAC TTC AGC CTG GAC ATC GAC GT - #G GGC TGC ACC GAC CTG         2592                                                                          His Ser His His Phe Ser Leu Asp Ile Asp Va - #l Gly Cys Thr Asp Leu           #   860                                                                       - CAC GAG AAC CTG GGC GTG TGG GTG GTG TTC AA - #G ATC AAG ACC CAG GAG         2640                                                                          His Glu Asn Leu Gly Val Trp Val Val Phe Ly - #s Ile Lys Thr Gln Glu           865                 8 - #70                 8 - #75                 8 -       #80                                                                           - GGC CAC GCC CGC CTG GGC AAC CTG GAG TTC AT - #C GAG GAG AAG CCC CTG         2688                                                                          Gly His Ala Arg Leu Gly Asn Leu Glu Phe Il - #e Glu Glu Lys Pro Leu           #               895                                                           - CTG GGC GAG GCC CTG AGC CGC GTG AAG CGC GC - #C GAG AAG AAG TGG CGC         2736                                                                          Leu Gly Glu Ala Leu Ser Arg Val Lys Arg Al - #a Glu Lys Lys Trp Arg           #           910                                                               - GAC AAG CGC GAG AAG CTG CAG CTG GAG ACC AA - #G CGC GTG TAC ACC GAG         2784                                                                          Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Ly - #s Arg Val Tyr Thr Glu           #       925                                                                   - GCC AAG GAG GCC GTG GAC GCC CTG TTC GTG GA - #C AGC CAG TAC GAC CGC         2832                                                                          Ala Lys Glu Ala Val Asp Ala Leu Phe Val As - #p Ser Gln Tyr Asp Arg           #   940                                                                       - CTG CAG GCC GAC ACC AAC ATC GGC ATG ATC CA - #C GCC GCC GAC AAG CTG         2880                                                                          Leu Gln Ala Asp Thr Asn Ile Gly Met Ile Hi - #s Ala Ala Asp Lys Leu           945                 9 - #50                 9 - #55                 9 -       #60                                                                           - GTG CAC CGC ATC CGC GAG GCC TAC CTG AGC GA - #G CTG CCC GTG ATC CCC         2928                                                                          Val His Arg Ile Arg Glu Ala Tyr Leu Ser Gl - #u Leu Pro Val Ile Pro           #               975                                                           - GGC GTG AAC GCC GAG ATC TTC GAG GAG CTG GA - #G GGC CAC ATC ATC ACC         2976                                                                          Gly Val Asn Ala Glu Ile Phe Glu Glu Leu Gl - #u Gly His Ile Ile Thr           #           990                                                               - GCC ATC AGC CTG TAC GAC GCC CGC AAC GTG GT - #G AAG AAC GGC GAC TTC         3024                                                                          Ala Ile Ser Leu Tyr Asp Ala Arg Asn Val Va - #l Lys Asn Gly Asp Phe           #      10050                                                                  - AAC AAC GGC CTG ACC TGC TGG AAC GTG AAG GG - #C CAC GTG GAC GTG CAG         3072                                                                          Asn Asn Gly Leu Thr Cys Trp Asn Val Lys Gl - #y His Val Asp Val Gln           #  10205                                                                      - CAG AGC CAC CAC CGC AGC GAC CTG GTG ATC CC - #C GAG TGG GAG GCC GAG         3120                                                                          Gln Ser His His Arg Ser Asp Leu Val Ile Pr - #o Glu Trp Glu Ala Glu           #               10401030 - #                1035                              - GTG AGC CAG GCC GTG CGC GTG TGC CCC GGC TG - #C GGC TAC ATC CTG CGC         3168                                                                          Val Ser Gln Ala Val Arg Val Cys Pro Gly Cy - #s Gly Tyr Ile Leu Arg           #              10550                                                          - GTG ACC GCC TAC AAG GAG GGC TAC GGC GAG GG - #C TGC GTG ACC ATC CAC         3216                                                                          Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gl - #y Cys Val Thr Ile His           #          10705                                                              - GAG ATC GAG AAC AAC ACC GAC GAG CTG AAG TT - #C AAG AAC CGC GAG GAG         3264                                                                          Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Ph - #e Lys Asn Arg Glu Glu           #      10850                                                                  - GAG GAG GTG TAC CCC ACC GAC ACC GGC ACC TG - #C AAC GAC TAC ACC GCC         3312                                                                          Glu Glu Val Tyr Pro Thr Asp Thr Gly Thr Cy - #s Asn Asp Tyr Thr Ala           #  11005                                                                      - CAC CAG GGC ACC GCC GGC TGC GCC GAC GCC TG - #C AAC AGC CGC AAC GCC         3360                                                                          His Gln Gly Thr Ala Gly Cys Ala Asp Ala Cy - #s Asn Ser Arg Asn Ala           #               11201110 - #                1115                              - GGC TAC GAG GAC GCC TAC GAG GTG GAC ACC AC - #C GCC AGC GTG AAC TAC         3408                                                                          Gly Tyr Glu Asp Ala Tyr Glu Val Asp Thr Th - #r Ala Ser Val Asn Tyr           #              11350                                                          - AAG CCC ACC TAC GAG GAG GAG ACC TAC ACC GA - #C GTG CGC CGC GAC AAC         3456                                                                          Lys Pro Thr Tyr Glu Glu Glu Thr Tyr Thr As - #p Val Arg Arg Asp Asn           #          11505                                                              - CAC TGC GAG TAC GAC CGC GGC TAC GTG AAC TA - #C CCC CCC GTG CCC GCC         3504                                                                          His Cys Glu Tyr Asp Arg Gly Tyr Val Asn Ty - #r Pro Pro Val Pro Ala           #      11650                                                                  - GGC TAC GTG ACC AAG GAG CTG GAG TAC TTC CC - #C GAG ACC GAC ACC GTG         3552                                                                          Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pr - #o Glu Thr Asp Thr Val           #  11805                                                                      - TGG ATC GAG ATC GGC GAG ACC GAG GGC AAG TT - #C ATC GTG GAC AGC GTG         3600                                                                          Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys Ph - #e Ile Val Asp Ser Val           #               12001190 - #                1195                              #              3624TG GAG GAG TAG                                             Glu Leu Leu Leu Met Glu Glu                                                                   1205                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1207 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Met Asp Leu Leu Pro Asp Ala Arg Ile Glu As - #p Ser Leu Cys Ile Ala         #                 15                                                          - Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Al - #a Ser Thr Val Gln Thr         #             30                                                              - Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Va - #l Leu Gly Val Pro Phe         #         45                                                                  - Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Le - #u Val Gly Glu Leu Trp         #     60                                                                      - Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe Le - #u Glu His Val Glu Gln         # 80                                                                          - Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Ar - #g Asn Thr Ala Leu Ala         #                 95                                                          - Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Al - #a Tyr Gln Gln Ser Leu         #           110                                                               - Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Ar - #g Thr Arg Ser Val Leu         #       125                                                                   - Tyr Thr Gln Tyr Ile Ala Leu Glu Leu Asp Ph - #e Leu Asn Ala Met Pro         #   140                                                                       - Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Le - #u Leu Met Val Tyr Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Ala Ala Asn Leu His Leu Leu Leu Leu Ar - #g Asp Ala Ser Leu Phe         #               175                                                           - Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Il - #e Gln Arg Tyr Tyr Glu         #           190                                                               - Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser As - #p Tyr Cys Val Glu Trp         #       205                                                                   - Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Th - #r Asn Ala Ala Ser Trp         #   220                                                                       - Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Th - #r Leu Gly Val Leu Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Ar - #g Thr Tyr Pro Ile Asn         #               255                                                           - Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Th - #r Asp Ala Ile Gly Ala         #           270                                                               - Thr Gly Val Asn Met Ala Ser Met Asn Trp Ty - #r Asn Asn Asn Ala Pro         #       285                                                                   - Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Ar - #g Ser Pro His Leu Leu         #   300                                                                       - Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Al - #a Ser Ser Arg Trp Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Thr Arg His Met Thr Tyr Trp Arg Gly Hi - #s Thr Ile Gln Ser Arg         #               335                                                           - Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr Hi - #s Gly Ala Thr Asn Thr         #           350                                                               - Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Se - #r Arg Asp Val Tyr Arg         #       365                                                                   - Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gl - #y Ile Tyr Leu Glu Pro         #   380                                                                       - Ile His Gly Val Pro Thr Val Arg Phe Asn Ph - #e Thr Asn Pro Gln Asn         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gl - #n Pro Tyr Glu Ser Pro         #               415                                                           - Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu Le - #u Pro Pro Glu Thr Thr         #           430                                                               - Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Ar - #g Leu Ser His Ile Gly         #       445                                                                   - Ile Ile Leu Gln Ser Arg Val Asn Val Pro Va - #l Tyr Ser Trp Thr His         #   460                                                                       - Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pr - #o Asn Arg Ile Thr Gln         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gl - #n Gly Thr Thr Val Val         #               495                                                           - Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Le - #u Arg Arg Thr Asn Thr         #           510                                                               - Gly Gly Phe Gly Pro Ile Arg Val Thr Val As - #n Gly Pro Leu Thr Gln         #       525                                                                   - Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Th - #r Val Asp Phe Asp Phe         #   540                                                                       - Phe Val Ser Arg Gly Gly Thr Thr Val Asn As - #n Phe Arg Phe Leu Arg         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gl - #y Asn Phe Val Arg Arg         #               575                                                           - Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Il - #e Gln Asp Ile Ile Arg         #           590                                                               - Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly Gl - #u Val Tyr Ile Asp Lys         #       605                                                                   - Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Gl - #u Ala Glu Tyr Asp Leu         #   620                                                                       - Glu Arg Ala Gln Glu Ala Val Asn Ala Leu Ph - #e Thr Asn Thr Asn Pro         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Arg Arg Leu Lys Thr Asp Val Thr Asp Tyr Hi - #s Ile Asp Gln Val Ser         #               655                                                           - Asn Leu Val Ala Cys Leu Ser Asp Glu Phe Cy - #s Leu Asp Glu Lys Arg         #           670                                                               - Glu Leu Leu Glu Lys Val Lys Tyr Ala Lys Ar - #g Leu Ser Asp Glu Arg         #       685                                                                   - Asn Leu Leu Gln Asp Pro Asn Phe Thr Ser Il - #e Asn Lys Gln Pro Asp         #   700                                                                       - Phe Ile Ser Thr Asn Glu Gln Ser Asn Phe Th - #r Ser Ile His Glu Gln         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Ser Glu His Gly Trp Trp Gly Ser Glu Asn Il - #e Thr Ile Gln Glu Gly         #               735                                                           - Asn Asp Val Phe Lys Glu Asn Tyr Val Thr Le - #u Pro Gly Thr Phe Asn         #           750                                                               - Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Il - #e Gly Glu Ser Glu Leu         #       765                                                                   - Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Ty - #r Ile Glu Asp Ser Gln         #   780                                                                       - Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Al - #a Lys His Glu Thr Leu         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asp Val Pro Gly Thr Glu Ser Leu Trp Pro Le - #u Ser Val Glu Ser Pro         #               815                                                           - Ile Gly Arg Cys Gly Glu Pro Asn Arg Cys Al - #a Pro His Phe Glu Trp         #           830                                                               - Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gl - #y Glu Lys Cys Ala His         #       845                                                                   - His Ser His His Phe Ser Leu Asp Ile Asp Va - #l Gly Cys Thr Asp Leu         #   860                                                                       - His Glu Asn Leu Gly Val Trp Val Val Phe Ly - #s Ile Lys Thr Gln Glu         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Gly His Ala Arg Leu Gly Asn Leu Glu Phe Il - #e Glu Glu Lys Pro Leu         #               895                                                           - Leu Gly Glu Ala Leu Ser Arg Val Lys Arg Al - #a Glu Lys Lys Trp Arg         #           910                                                               - Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Ly - #s Arg Val Tyr Thr Glu         #       925                                                                   - Ala Lys Glu Ala Val Asp Ala Leu Phe Val As - #p Ser Gln Tyr Asp Arg         #   940                                                                       - Leu Gln Ala Asp Thr Asn Ile Gly Met Ile Hi - #s Ala Ala Asp Lys Leu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Val His Arg Ile Arg Glu Ala Tyr Leu Ser Gl - #u Leu Pro Val Ile Pro         #               975                                                           - Gly Val Asn Ala Glu Ile Phe Glu Glu Leu Gl - #u Gly His Ile Ile Thr         #           990                                                               - Ala Ile Ser Leu Tyr Asp Ala Arg Asn Val Va - #l Lys Asn Gly Asp Phe         #      10050                                                                  - Asn Asn Gly Leu Thr Cys Trp Asn Val Lys Gl - #y His Val Asp Val Gln         #  10205                                                                      - Gln Ser His His Arg Ser Asp Leu Val Ile Pr - #o Glu Trp Glu Ala Glu         #               10401030 - #                1035                              - Val Ser Gln Ala Val Arg Val Cys Pro Gly Cy - #s Gly Tyr Ile Leu Arg         #              10550                                                          - Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gl - #y Cys Val Thr Ile His         #          10705                                                              - Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Ph - #e Lys Asn Arg Glu Glu         #      10850                                                                  - Glu Glu Val Tyr Pro Thr Asp Thr Gly Thr Cy - #s Asn Asp Tyr Thr Ala         #  11005                                                                      - His Gln Gly Thr Ala Gly Cys Ala Asp Ala Cy - #s Asn Ser Arg Asn Ala         #               11201110 - #                1115                              - Gly Tyr Glu Asp Ala Tyr Glu Val Asp Thr Th - #r Ala Ser Val Asn Tyr         #              11350                                                          - Lys Pro Thr Tyr Glu Glu Glu Thr Tyr Thr As - #p Val Arg Arg Asp Asn         #          11505                                                              - His Cys Glu Tyr Asp Arg Gly Tyr Val Asn Ty - #r Pro Pro Val Pro Ala         #      11650                                                                  - Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pr - #o Glu Thr Asp Thr Val         #  11805                                                                      - Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys Ph - #e Ile Val Asp Ser Val         #               12001190 - #                1195                              - Glu Leu Leu Leu Met Glu Glu                                                                 1205                                                          - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3468 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3465                                               #/product= "Full-length, hybrid,                                              #maize optimized cryIA(b)"                                                    #"Disclosed in Figure 7 as contained in pCIB4434 - #."                        -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - ATG GAC AAC AAC CCC AAC ATC AAC GAG TGC AT - #C CCC TAC AAC TGC CTG           48                                                                          Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu           #      12205                                                                  - AGC AAC CCC GAG GTG GAG GTG CTG GGC GGC GA - #G CGC ATC GAG ACC GGC           96                                                                          Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly           #  12350                                                                      - TAC ACC CCC ATC GAC ATC AGC CTG AGC CTG AC - #C CAG TTC CTG CTG AGC          144                                                                          Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser           #               12551245 - #                1250                              - GAG TTC GTG CCC GGC GCC GGC TTC GTG CTG GG - #C CTG GTG GAC ATC ATC          192                                                                          Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           #              12705                                                          - TGG GGC ATC TTC GGC CCC AGC CAG TGG GAC GC - #C TTC CTG GTG CAG ATC          240                                                                          Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile           #          12850                                                              - GAG CAG CTG ATC AAC CAG CGC ATC GAG GAG TT - #C GCC CGC AAC CAG GCC          288                                                                          Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala           #      13005                                                                  - ATC AGC CGC CTG GAG GGC CTG AGC AAC CTG TA - #C CAA ATC TAC GCC GAG          336                                                                          Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu           #  13150                                                                      - AGC TTC CGC GAG TGG GAG GCC GAC CCC ACC AA - #C CCC GCC CTG CGC GAG          384                                                                          Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu           #               13351325 - #                1330                              - GAG ATG CGC ATC CAG TTC AAC GAC ATG AAC AG - #C GCC CTG ACC ACC GCC          432                                                                          Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala           #              13505                                                          - ATC CCC CTG TTC GCC GTG CAG AAC TAC CAG GT - #G CCC CTG CTG AGC GTG          480                                                                          Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           #          13650                                                              - TAC GTG CAG GCC GCC AAC CTG CAC CTG AGC GT - #G CTG CGC GAC GTC AGC          528                                                                          Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser           #      13805                                                                  - GTG TTC GGC CAG CGC TGG GGC TTC GAC GCC GC - #C ACC ATC AAC AGC CGC          576                                                                          Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg           #  13950                                                                      - TAC AAC GAC CTG ACC CGC CTG ATC GGC AAC TA - #C ACC GAC CAC GCC GTG          624                                                                          Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val           #               14151405 - #                1410                              - CGC TGG TAC AAC ACC GGC CTG GAG CGC GTG TG - #G GGT CCC GAC AGC CGC          672                                                                          Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg           #              14305                                                          - GAC TGG ATC AGG TAC AAC CAG TTC CGC CGC GA - #G CTG ACC CTG ACC GTG          720                                                                          Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           #          14450                                                              - CTG GAC ATC GTG AGC CTG TTC CCC AAC TAC GA - #C AGC CGC ACC TAC CCC          768                                                                          Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro           #      14605                                                                  - ATC CGC ACC GTG AGC CAG CTG ACC CGC GAG AT - #T TAC ACC AAC CCC GTG          816                                                                          Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val           #  14750                                                                      - CTG GAG AAC TTC GAC GGC AGC TTC CGC GGC AG - #C GCC CAG GGC ATC GAG          864                                                                          Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu           #               14951485 - #                1490                              - GGC AGC ATC CGC AGC CCC CAC CTG ATG GAC AT - #C CTG AAC AGC ATC ACC          912                                                                          Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr           #              15105                                                          - ATC TAC ACC GAC GCC CAC CGC GGC GAG TAC TA - #C TGG AGC GGC CAC CAG          960                                                                          Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln           #          15250                                                              - ATC ATG GCC AGC CCC GTC GGC TTC AGC GGC CC - #C GAG TTC ACC TTC CCC         1008                                                                          Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro           #      15405                                                                  - CTG TAC GGC ACC ATG GGC AAC GCT GCA CCT CA - #G CAG CGC ATC GTG GCA         1056                                                                          Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala           #  15550                                                                      - CAG CTG GGC CAG GGA GTG TAC CGC ACC CTG AG - #C AGC ACC CTG TAC CGT         1104                                                                          Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg           #               15751565 - #                1570                              - CGA CCT TTC AAC ATC GGC ATC AAC AAC CAG CA - #G CTG AGC GTG CTG GAC         1152                                                                          Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp           #              15905                                                          - GGC ACC GAG TTC GCC TAC GGC ACC AGC AGC AA - #C CTG CCC AGC GCC GTG         1200                                                                          Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           #          16050                                                              - TAC CGC AAG AGC GGC ACC GTG GAC AGC CTG GA - #C GAG ATC CCC CCT CAG         1248                                                                          Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln           #      16205                                                                  - AAC AAC AAC GTG CCA CCT CGA CAG GGC TTC AG - #C CAC CGT CTG AGC CAC         1296                                                                          Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His           #  16350                                                                      - GTG AGC ATG TTC CGC AGT GGC TTC AGC AAC AG - #C AGC GTG AGC ATC ATC         1344                                                                          Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile           #               16551645 - #                1650                              - CGT GCA CCT ATG TTC AGC TGG ATT CAC CGC AG - #T GCC GAG TTC AAC AAC         1392                                                                          Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn           #              16705                                                          - ATC ATC CCC AGC AGC CAG ATC ACC CAG ATC CC - #C CTG ACC AAG AGC ACC         1440                                                                          Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           #          16850                                                              - AAC CTG GGC AGC GGC ACC AGC GTG GTG AAG GG - #C CCC GGC TTC ACC GGC         1488                                                                          Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly           #      17005                                                                  - GGC GAC ATC CTG CGC CGC ACC AGC CCC GGC CA - #G ATC AGC ACC CTG CGC         1536                                                                          Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg           #  17150                                                                      - GTG AAC ATC ACC GCC CCC CTG AGC CAG CGC TA - #C CGC GTC CGC ATC CGC         1584                                                                          Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg           #               17351725 - #                1730                              - TAC GCC AGC ACC ACC AAC CTG CAG TTC CAC AC - #C AGC ATC GAC GGC CGC         1632                                                                          Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg           #              17505                                                          - CCC ATC AAC CAG GGC AAC TTC AGC GCC ACC AT - #G AGC AGC GGC AGC AAC         1680                                                                          Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           #          17650                                                              - CTG CAG AGC GGC AGC TTC CGC ACC GTG GGC TT - #C ACC ACC CCC TTC AAC         1728                                                                          Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn           #      17805                                                                  - TTC AGC AAC GGC AGC AGC GTG TTC ACC CTG AG - #C GCC CAC GTG TTC AAC         1776                                                                          Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn           #  17950                                                                      - AGC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTG CCC GCC GAG         1824                                                                          Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu           #               18151805 - #                1810                              - GTG ACC TTC GAG GCC GAG TAC GAC CTG GAG AG - #G GCT CAG AAG GCC GTG         1872                                                                          Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val           #              18305                                                          - AAC GAG CTG TTC ACC AGC AGC AAC CAG ATC GG - #C CTG AAG ACC GAC GTG         1920                                                                          Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val           #          18450                                                              - ACC GAC TAC CAC ATC GAT CAA GTA TCC AAT TT - #A GTT GAG TGT TTA TCT         1968                                                                          Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser           #      18605                                                                  - GAT GAA TTT TGT CTG GAT GAA AAA AAA GAA TT - #G TCC GAG AAA GTC AAA         2016                                                                          Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys           #  18750                                                                      - CAT GCG AAG CGA CTT AGT GAT GAG CGG AAT TT - #A CTT CAA GAT CCA AAC         2064                                                                          His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn           #               18951885 - #                1890                              - TTT AGA GGG ATC AAT AGA CAA CTA GAC CGT GG - #C TGG AGA GGA AGT ACG         2112                                                                          Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr           #              19105                                                          - GAT ATT ACC ATC CAA GGA GGC GAT GAC GTA TT - #C AAA GAG AAT TAC GTT         2160                                                                          Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val           #          19250                                                              - ACG CTA TTG GGT ACC TTT GAT GAG TGC TAT CC - #A ACG TAT TTA TAT CAA         2208                                                                          Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln           #      19405                                                                  - AAA ATA GAT GAG TCG AAA TTA AAA GCC TAT AC - #C CGT TAC CAA TTA AGA         2256                                                                          Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg           #  19550                                                                      - GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA AT - #C TAT TTA ATT CGC TAC         2304                                                                          Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr           #               19751965 - #                1970                              - AAT GCC AAA CAC GAA ACA GTA AAT GTG CCA GG - #T ACG GGT TCC TTA TGG         2352                                                                          Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp           #              19905                                                          - CCG CTT TCA GCC CCA AGT CCA ATC GGA AAA TG - #T GCC CAT CAT TCC CAT         2400                                                                          Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Ala His His Ser His           #          20050                                                              - CAT TTC TCC TTG GAC ATT GAT GTT GGA TGT AC - #A GAC TTA AAT GAG GAC         2448                                                                          His Phe Ser Leu Asp Ile Asp Val Gly Cys Th - #r Asp Leu Asn Glu Asp           #      20205                                                                  - TTA GGT GTA TGG GTG ATA TTC AAG ATT AAG AC - #G CAA GAT GGC CAT GCA         2496                                                                          Leu Gly Val Trp Val Ile Phe Lys Ile Lys Th - #r Gln Asp Gly His Ala           #  20350                                                                      - AGA CTA GGA AAT CTA GAA TTT CTC GAA GAG AA - #A CCA TTA GTA GGA GAA         2544                                                                          Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Ly - #s Pro Leu Val Gly Glu           #               20552045 - #                2050                              - GCA CTA GCT CGT GTG AAA AGA GCG GAG AAA AA - #A TGG AGA GAC AAA CGT         2592                                                                          Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Ly - #s Trp Arg Asp Lys Arg           #              20705                                                          - GAA AAA TTG GAA TGG GAA ACA AAT ATT GTT TA - #T AAA GAG GCA AAA GAA         2640                                                                          Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Ty - #r Lys Glu Ala Lys Glu           #          20850                                                              - TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA TA - #T GAT AGA TTA CAA GCG         2688                                                                          Ser Val Asp Ala Leu Phe Val Asn Ser Gln Ty - #r Asp Arg Leu Gln Ala           #      21005                                                                  - GAT ACC AAC ATC GCG ATG ATT CAT GCG GCA GA - #T AAA CGC GTT CAT AGC         2736                                                                          Asp Thr Asn Ile Ala Met Ile His Ala Ala As - #p Lys Arg Val His Ser           #  21150                                                                      - ATT CGA GAA GCT TAT CTG CCT GAG CTG TCT GT - #G ATT CCG GGT GTC AAT         2784                                                                          Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Va - #l Ile Pro Gly Val Asn           #               21352125 - #                2130                              - GCG GCT ATT TTT GAA GAA TTA GAA GGG CGT AT - #T TTC ACT GCA TTC TCC         2832                                                                          Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Il - #e Phe Thr Ala Phe Ser           #              21505                                                          - CTA TAT GAT GCG AGA AAT GTC ATT AAA AAT GG - #T GAT TTT AAT AAT GGC         2880                                                                          Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gl - #y Asp Phe Asn Asn Gly           #          21650                                                              - TTA TCC TGC TGG AAC GTG AAA GGG CAT GTA GA - #T GTA GAA GAA CAA AAC         2928                                                                          Leu Ser Cys Trp Asn Val Lys Gly His Val As - #p Val Glu Glu Gln Asn           #      21805                                                                  - AAC CAC CGT TCG GTC CTT GTT GTT CCG GAA TG - #G GAA GCA GAA GTG TCA         2976                                                                          Asn His Arg Ser Val Leu Val Val Pro Glu Tr - #p Glu Ala Glu Val Ser           #  21950                                                                      - CAA GAA GTT CGT GTC TGT CCG GGT CGT GGC TA - #T ATC CTT CGT GTC ACA         3024                                                                          Gln Glu Val Arg Val Cys Pro Gly Arg Gly Ty - #r Ile Leu Arg Val Thr           #               22152205 - #                2210                              - GCG TAC AAG GAG GGA TAT GGA GAA GGT TGC GT - #A ACC ATT CAT GAG ATC         3072                                                                          Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Va - #l Thr Ile His Glu Ile           #              22305                                                          - GAG AAC AAT ACA GAC GAA CTG AAG TTT AGC AA - #C TGT GTA GAA GAG GAA         3120                                                                          Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser As - #n Cys Val Glu Glu Glu           #          22450                                                              - GTA TAT CCA AAC AAC ACG GTA ACG TGT AAT GA - #T TAT ACT GCG ACT CAA         3168                                                                          Val Tyr Pro Asn Asn Thr Val Thr Cys Asn As - #p Tyr Thr Ala Thr Gln           #      22605                                                                  - GAA GAA TAT GAG GGT ACG TAC ACT TCT CGT AA - #T CGA GGA TAT GAC GGA         3216                                                                          Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg As - #n Arg Gly Tyr Asp Gly           #  22750                                                                      - GCC TAT GAA AGC AAT TCT TCT GTA CCA GCT GA - #T TAT GCA TCA GCC TAT         3264                                                                          Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala As - #p Tyr Ala Ser Ala Tyr           #               22952285 - #                2290                              - GAA GAA AAA GCA TAT ACA GAT GGA CGA AGA GA - #C AAT CCT TGT GAA TCT         3312                                                                          Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg As - #p Asn Pro Cys Glu Ser           #              23105                                                          - AAC AGA GGA TAT GGG GAT TAC ACA CCA CTA CC - #A GCT GGC TAT GTG ACA         3360                                                                          Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pr - #o Ala Gly Tyr Val Thr           #          23250                                                              - AAA GAA TTA GAG TAC TTC CCA GAA ACC GAT AA - #G GTA TGG ATT GAG ATC         3408                                                                          Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Ly - #s Val Trp Ile Glu Ile           #      23405                                                                  - GGA GAA ACG GAA GGA ACA TTC ATC GTG GAC AG - #C GTG GAA TTA CTT CTT         3456                                                                          Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Se - #r Val Glu Leu Leu Leu           #  23550                                                                      #     3468                                                                    Met Glu Glu                                                                   2360                                                                          - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1155 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu         #                 15                                                          - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly         #             30                                                              - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser         #         45                                                                  - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile         #     60                                                                      - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile         # 80                                                                          - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala         #                 95                                                          - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu         #           110                                                               - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu         #       125                                                                   - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala         #   140                                                                       - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser         #               175                                                           - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg         #           190                                                               - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val         #       205                                                                   - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg         #   220                                                                       - Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro         #               255                                                           - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val         #           270                                                               - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu         #       285                                                                   - Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr         #   300                                                                       - Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro         #               335                                                           - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala         #           350                                                               - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg         #       365                                                                   - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp         #   380                                                                       - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln         #               415                                                           - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His         #           430                                                               - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile         #       445                                                                   - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn         #   460                                                                       - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly         #               495                                                           - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg         #           510                                                               - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg         #       525                                                                   - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg         #   540                                                                       - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn         #               575                                                           - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn         #           590                                                               - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu         #       605                                                                   - Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val         #   620                                                                       - Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser         #               655                                                           - Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys         #           670                                                               - His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn         #       685                                                                   - Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr         #   700                                                                       - Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln         #               735                                                           - Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg         #           750                                                               - Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr         #       765                                                                   - Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp         #   780                                                                       - Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Ala His His Ser His         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - His Phe Ser Leu Asp Ile Asp Val Gly Cys Th - #r Asp Leu Asn Glu Asp         #               815                                                           - Leu Gly Val Trp Val Ile Phe Lys Ile Lys Th - #r Gln Asp Gly His Ala         #           830                                                               - Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Ly - #s Pro Leu Val Gly Glu         #       845                                                                   - Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Ly - #s Trp Arg Asp Lys Arg         #   860                                                                       - Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Ty - #r Lys Glu Ala Lys Glu         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Ser Val Asp Ala Leu Phe Val Asn Ser Gln Ty - #r Asp Arg Leu Gln Ala         #               895                                                           - Asp Thr Asn Ile Ala Met Ile His Ala Ala As - #p Lys Arg Val His Ser         #           910                                                               - Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Va - #l Ile Pro Gly Val Asn         #       925                                                                   - Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Il - #e Phe Thr Ala Phe Ser         #   940                                                                       - Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gl - #y Asp Phe Asn Asn Gly         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Leu Ser Cys Trp Asn Val Lys Gly His Val As - #p Val Glu Glu Gln Asn         #               975                                                           - Asn His Arg Ser Val Leu Val Val Pro Glu Tr - #p Glu Ala Glu Val Ser         #           990                                                               - Gln Glu Val Arg Val Cys Pro Gly Arg Gly Ty - #r Ile Leu Arg Val Thr         #      10050                                                                  - Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Va - #l Thr Ile His Glu Ile         #  10205                                                                      - Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser As - #n Cys Val Glu Glu Glu         #               10401030 - #                1035                              - Val Tyr Pro Asn Asn Thr Val Thr Cys Asn As - #p Tyr Thr Ala Thr Gln         #              10550                                                          - Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg As - #n Arg Gly Tyr Asp Gly         #          10705                                                              - Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala As - #p Tyr Ala Ser Ala Tyr         #      10850                                                                  - Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg As - #p Asn Pro Cys Glu Ser         #  11005                                                                      - Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pr - #o Ala Gly Tyr Val Thr         #               11201110 - #                1115                              - Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Ly - #s Val Trp Ile Glu Ile         #              11350                                                          - Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Se - #r Val Glu Leu Leu Leu         #          11505                                                              - Met Glu Glu                                                                         1155                                                                  - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3546 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3543                                               #/product= "Full-length, hybrid,                                                             maize opt - #imized heat stable cryIA(b)"                      #"Disclosed in Figure 9 as contained in pCIB5511 - #."                        -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - ATG GAC AAC AAC CCC AAC ATC AAC GAG TGC AT - #C CCC TAC AAC TGC CTG           48                                                                          Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu           #              11705                                                          - AGC AAC CCC GAG GTG GAG GTG CTG GGC GGC GA - #G CGC ATC GAG ACC GGC           96                                                                          Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly           #          11850                                                              - TAC ACC CCC ATC GAC ATC AGC CTG AGC CTG AC - #C CAG TTC CTG CTG AGC          144                                                                          Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser           #      12005                                                                  - GAG TTC GTG CCC GGC GCC GGC TTC GTG CTG GG - #C CTG GTG GAC ATC ATC          192                                                                          Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           #  12150                                                                      - TGG GGC ATC TTC GGC CCC AGC CAG TGG GAC GC - #C TTC CTG GTG CAG ATC          240                                                                          Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile           #               12351225 - #                1230                              - GAG CAG CTG ATC AAC CAG CGC ATC GAG GAG TT - #C GCC CGC AAC CAG GCC          288                                                                          Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala           #              12505                                                          - ATC AGC CGC CTG GAG GGC CTG AGC AAC CTG TA - #C CAA ATC TAC GCC GAG          336                                                                          Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu           #          12650                                                              - AGC TTC CGC GAG TGG GAG GCC GAC CCC ACC AA - #C CCC GCC CTG CGC GAG          384                                                                          Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu           #      12805                                                                  - GAG ATG CGC ATC CAG TTC AAC GAC ATG AAC AG - #C GCC CTG ACC ACC GCC          432                                                                          Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala           #  12950                                                                      - ATC CCC CTG TTC GCC GTG CAG AAC TAC CAG GT - #G CCC CTG CTG AGC GTG          480                                                                          Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           #               13151305 - #                1310                              - TAC GTG CAG GCC GCC AAC CTG CAC CTG AGC GT - #G CTG CGC GAC GTC AGC          528                                                                          Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser           #              13305                                                          - GTG TTC GGC CAG CGC TGG GGC TTC GAC GCC GC - #C ACC ATC AAC AGC CGC          576                                                                          Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg           #          13450                                                              - TAC AAC GAC CTG ACC CGC CTG ATC GGC AAC TA - #C ACC GAC CAC GCC GTG          624                                                                          Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val           #      13605                                                                  - CGC TGG TAC AAC ACC GGC CTG GAG CGC GTG TG - #G GGT CCC GAC AGC CGC          672                                                                          Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg           #  13750                                                                      - GAC TGG ATC AGG TAC AAC CAG TTC CGC CGC GA - #G CTG ACC CTG ACC GTG          720                                                                          Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           #               13951385 - #                1390                              - CTG GAC ATC GTG AGC CTG TTC CCC AAC TAC GA - #C AGC CGC ACC TAC CCC          768                                                                          Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro           #              14105                                                          - ATC CGC ACC GTG AGC CAG CTG ACC CGC GAG AT - #T TAC ACC AAC CCC GTG          816                                                                          Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val           #          14250                                                              - CTG GAG AAC TTC GAC GGC AGC TTC CGC GGC AG - #C GCC CAG GGC ATC GAG          864                                                                          Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu           #      14405                                                                  - GGC AGC ATC CGC AGC CCC CAC CTG ATG GAC AT - #C CTG AAC AGC ATC ACC          912                                                                          Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr           #  14550                                                                      - ATC TAC ACC GAC GCC CAC CGC GGC GAG TAC TA - #C TGG AGC GGC CAC CAG          960                                                                          Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln           #               14751465 - #                1470                              - ATC ATG GCC AGC CCC GTC GGC TTC AGC GGC CC - #C GAG TTC ACC TTC CCC         1008                                                                          Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro           #              14905                                                          - CTG TAC GGC ACC ATG GGC AAC GCT GCA CCT CA - #G CAG CGC ATC GTG GCA         1056                                                                          Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala           #          15050                                                              - CAG CTG GGC CAG GGA GTG TAC CGC ACC CTG AG - #C AGC ACC CTG TAC CGT         1104                                                                          Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg           #      15205                                                                  - CGA CCT TTC AAC ATC GGC ATC AAC AAC CAG CA - #G CTG AGC GTG CTG GAC         1152                                                                          Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp           #  15350                                                                      - GGC ACC GAG TTC GCC TAC GGC ACC AGC AGC AA - #C CTG CCC AGC GCC GTG         1200                                                                          Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           #               15551545 - #                1550                              - TAC CGC AAG AGC GGC ACC GTG GAC AGC CTG GA - #C GAG ATC CCC CCT CAG         1248                                                                          Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln           #              15705                                                          - AAC AAC AAC GTG CCA CCT CGA CAG GGC TTC AG - #C CAC CGT CTG AGC CAC         1296                                                                          Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His           #          15850                                                              - GTG AGC ATG TTC CGC AGT GGC TTC AGC AAC AG - #C AGC GTG AGC ATC ATC         1344                                                                          Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile           #      16005                                                                  - CGT GCA CCT ATG TTC AGC TGG ATT CAC CGC AG - #T GCC GAG TTC AAC AAC         1392                                                                          Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn           #  16150                                                                      - ATC ATC CCC AGC AGC CAG ATC ACC CAG ATC CC - #C CTG ACC AAG AGC ACC         1440                                                                          Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           #               16351625 - #                1630                              - AAC CTG GGC AGC GGC ACC AGC GTG GTG AAG GG - #C CCC GGC TTC ACC GGC         1488                                                                          Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly           #              16505                                                          - GGC GAC ATC CTG CGC CGC ACC AGC CCC GGC CA - #G ATC AGC ACC CTG CGC         1536                                                                          Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg           #          16650                                                              - GTG AAC ATC ACC GCC CCC CTG AGC CAG CGC TA - #C CGC GTC CGC ATC CGC         1584                                                                          Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg           #      16805                                                                  - TAC GCC AGC ACC ACC AAC CTG CAG TTC CAC AC - #C AGC ATC GAC GGC CGC         1632                                                                          Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg           #  16950                                                                      - CCC ATC AAC CAG GGC AAC TTC AGC GCC ACC AT - #G AGC AGC GGC AGC AAC         1680                                                                          Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           #               17151705 - #                1710                              - CTG CAG AGC GGC AGC TTC CGC ACC GTG GGC TT - #C ACC ACC CCC TTC AAC         1728                                                                          Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn           #              17305                                                          - TTC AGC AAC GGC AGC AGC GTG TTC ACC CTG AG - #C GCC CAC GTG TTC AAC         1776                                                                          Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn           #          17450                                                              - AGC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTG CCC GCC GAG         1824                                                                          Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu           #      17605                                                                  - GTG ACC TTC GAG GCC GAG TAC GAC CTG GAG AG - #G GCT CAG AAG GCC GTG         1872                                                                          Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val           #  17750                                                                      - AAC GAG CTG TTC ACC AGC AGC AAC CAG ATC GG - #C CTG AAG ACC GAC GTG         1920                                                                          Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val           #               17951785 - #                1790                              - ACC GAC TAC CAC ATC GAT CAA GTA TCC AAT TT - #A GTT GAG TGT TTA TCT         1968                                                                          Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser           #              18105                                                          - GAT GAA TTT TGT CTG GAT GAA AAA AAA GAA TT - #G TCC GAG AAA GTC AAA         2016                                                                          Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys           #          18250                                                              - CAT GCG AAG CGA CTT AGT GAT GAG CGG AAT TT - #A CTT CAA GAT CCA AAC         2064                                                                          His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn           #      18405                                                                  - TTT AGA GGG ATC AAT AGA CAA CTA GAC CGT GG - #C TGG AGA GGA AGT ACG         2112                                                                          Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr           #  18550                                                                      - GAT ATT ACC ATC CAA GGA GGC GAT GAC GTA TT - #C AAA GAG AAT TAC GTT         2160                                                                          Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val           #               18751865 - #                1870                              - ACG CTA TTG GGT ACC TTC GAC GAG TGC TAC CC - #C ACC TAC CTG TAC CAG         2208                                                                          Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln           #              18905                                                          - AAG ATC GAC GAG AGC AAG CTG AAG GCC TAC AC - #C CGC TAC CAG CTG CGC         2256                                                                          Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg           #          19050                                                              - GGC TAC ATC GAG GAC AGC CAG GAC CTG GAA AT - #C TAC CTG ATC CGC TAC         2304                                                                          Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr           #      19205                                                                  - AAC GCC AAG CAC GAG ACC GTG AAC GTG CCC GG - #C ACC GGC AGC CTG TGG         2352                                                                          Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp           #  19350                                                                      - CCC CTG AGC GCC CCC AGC CCC ATC GGC AAG TG - #C GGG GAG CCG AAT CGA         2400                                                                          Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg           #               19551945 - #                1950                              - TGC GCT CCG CAC CTG GAG TGG AAC CCG GAC CT - #A GAC TGC AGC TGC AGG         2448                                                                          Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg           #              19705                                                          - GAC GGG GAG AAG TGC GCC CAC CAC AGC CAC CA - #C TTC AGC CTG GAC ATC         2496                                                                          Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile           #          19850                                                              - GAC GTG GGC TGC ACC GAC CTG AAC GAG GAC CT - #G GGC GTG TGG GTG ATC         2544                                                                          Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile           #      20005                                                                  - TTC AAG ATC AAG ACC CAG GAC GGC CAC GCC CG - #C CTG GGC AAT CTA GAA         2592                                                                          Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu           #  20150                                                                      - TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GC - #A CTA GCT CGT GTG AAA         2640                                                                          Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys           #               20352025 - #                2030                              - AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GA - #A AAA TTG GAA TGG GAA         2688                                                                          Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu           #              20505                                                          - ACA AAT ATT GTT TAT AAA GAG GCA AAA GAA TC - #T GTA GAT GCT TTA TTT         2736                                                                          Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe           #          20650                                                              - GTA AAC TCT CAA TAT GAT AGA TTA CAA GCG GA - #T ACC AAC ATC GCG ATG         2784                                                                          Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met           #      20805                                                                  - ATT CAT GCG GCA GAT AAA CGC GTT CAT AGC AT - #T CGA GAA GCT TAT CTG         2832                                                                          Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu           #  20950                                                                      - CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GC - #G GCT ATT TTT GAA GAA         2880                                                                          Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu           #               21152105 - #                2110                              - TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CT - #A TAT GAT GCG AGA AAT         2928                                                                          Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn           #              21305                                                          - GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TT - #A TCC TGC TGG AAC GTG         2976                                                                          Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val           #          21450                                                              - AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AA - #C CAC CGT TCG GTC CTT         3024                                                                          Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu           #      21605                                                                  - GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CA - #A GAA GTT CGT GTC TGT         3072                                                                          Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys           #  21750                                                                      - CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GC - #G TAC AAG GAG GGA TAT         3120                                                                          Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr           #               21952185 - #                2190                              - GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GA - #G AAC AAT ACA GAC GAA         3168                                                                          Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu           #              22105                                                          - CTG AAG TTT AGC AAC TGT GTA GAA GAG GAA GT - #A TAT CCA AAC AAC ACG         3216                                                                          Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr           #          22250                                                              - GTA ACG TGT AAT GAT TAT ACT GCG ACT CAA GA - #A GAA TAT GAG GGT ACG         3264                                                                          Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr           #      22405                                                                  - TAC ACT TCT CGT AAT CGA GGA TAT GAC GGA GC - #C TAT GAA AGC AAT TCT         3312                                                                          Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser           #  22550                                                                      - TCT GTA CCA GCT GAT TAT GCA TCA GCC TAT GA - #A GAA AAA GCA TAT ACA         3360                                                                          Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr           #               22752265 - #                2270                              - GAT GGA CGA AGA GAC AAT CCT TGT GAA TCT AA - #C AGA GGA TAT GGG GAT         3408                                                                          Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp           #              22905                                                          - TAC ACA CCA CTA CCA GCT GGC TAT GTG ACA AA - #A GAA TTA GAG TAC TTC         3456                                                                          Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe           #          23050                                                              - CCA GAA ACC GAT AAG GTA TGG ATT GAG ATC GG - #A GAA ACG GAA GGA ACA         3504                                                                          Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr           #      23205                                                                  - TTC ATC GTG GAC AGC GTG GAA TTA CTT CTT AT - #G GAG GAA TAA                 #3546                                                                         Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                       #  23350                                                                      - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1181 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu         #                 15                                                          - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly         #             30                                                              - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser         #         45                                                                  - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile         #     60                                                                      - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile         # 80                                                                          - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala         #                 95                                                          - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu         #           110                                                               - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu         #       125                                                                   - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala         #   140                                                                       - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser         #               175                                                           - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg         #           190                                                               - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val         #       205                                                                   - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg         #   220                                                                       - Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro         #               255                                                           - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val         #           270                                                               - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu         #       285                                                                   - Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr         #   300                                                                       - Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro         #               335                                                           - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala         #           350                                                               - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg         #       365                                                                   - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp         #   380                                                                       - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln         #               415                                                           - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His         #           430                                                               - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile         #       445                                                                   - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn         #   460                                                                       - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly         #               495                                                           - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg         #           510                                                               - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg         #       525                                                                   - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg         #   540                                                                       - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn         #               575                                                           - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn         #           590                                                               - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu         #       605                                                                   - Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val         #   620                                                                       - Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser         #               655                                                           - Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys         #           670                                                               - His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn         #       685                                                                   - Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr         #   700                                                                       - Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln         #               735                                                           - Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg         #           750                                                               - Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr         #       765                                                                   - Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp         #   780                                                                       - Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg         #               815                                                           - Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile         #           830                                                               - Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile         #       845                                                                   - Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu         #   860                                                                       - Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu         #               895                                                           - Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe         #           910                                                               - Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met         #       925                                                                   - Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu         #   940                                                                       - Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn         #               975                                                           - Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val         #           990                                                               - Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu         #      10050                                                                  - Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys         #  10205                                                                      - Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr         #               10401030 - #                1035                              - Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu         #              10550                                                          - Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr         #          10705                                                              - Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr         #      10850                                                                  - Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser         #  11005                                                                      - Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr         #               11201110 - #                1115                              - Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp         #              11350                                                          - Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe         #          11505                                                              - Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr         #      11650                                                                  - Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                     #  11805                                                                      - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3546 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3543                                               #/product= "Full-length, hybrid,                                                             maize opt - #imized heat stable cryIA(b)"                      #"Disclosed in Figure 11 as contained in pCIB551 - #2"                        -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - ATG GAC AAC AAC CCC AAC ATC AAC GAG TGC AT - #C CCC TAC AAC TGC CTG           48                                                                          Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu           #          11950                                                              - AGC AAC CCC GAG GTG GAG GTG CTG GGC GGC GA - #G CGC ATC GAG ACC GGC           96                                                                          Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly           #      12105                                                                  - TAC ACC CCC ATC GAC ATC AGC CTG AGC CTG AC - #C CAG TTC CTG CTG AGC          144                                                                          Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser           #  12250                                                                      - GAG TTC GTG CCC GGC GCC GGC TTC GTG CTG GG - #C CTG GTG GAC ATC ATC          192                                                                          Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           #               12451235 - #                1240                              - TGG GGC ATC TTC GGC CCC AGC CAG TGG GAC GC - #C TTC CTG GTG CAG ATC          240                                                                          Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile           #              12605                                                          - GAG CAG CTG ATC AAC CAG CGC ATC GAG GAG TT - #C GCC CGC AAC CAG GCC          288                                                                          Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala           #          12750                                                              - ATC AGC CGC CTG GAG GGC CTG AGC AAC CTG TA - #C CAA ATC TAC GCC GAG          336                                                                          Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu           #      12905                                                                  - AGC TTC CGC GAG TGG GAG GCC GAC CCC ACC AA - #C CCC GCC CTG CGC GAG          384                                                                          Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu           #  13050                                                                      - GAG ATG CGC ATC CAG TTC AAC GAC ATG AAC AG - #C GCC CTG ACC ACC GCC          432                                                                          Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala           #               13251315 - #                1320                              - ATC CCC CTG TTC GCC GTG CAG AAC TAC CAG GT - #G CCC CTG CTG AGC GTG          480                                                                          Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           #              13405                                                          - TAC GTG CAG GCC GCC AAC CTG CAC CTG AGC GT - #G CTG CGC GAC GTC AGC          528                                                                          Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser           #          13550                                                              - GTG TTC GGC CAG CGC TGG GGC TTC GAC GCC GC - #C ACC ATC AAC AGC CGC          576                                                                          Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg           #      13705                                                                  - TAC AAC GAC CTG ACC CGC CTG ATC GGC AAC TA - #C ACC GAC CAC GCC GTG          624                                                                          Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val           #  13850                                                                      - CGC TGG TAC AAC ACC GGC CTG GAG CGC GTG TG - #G GGT CCC GAC AGC CGC          672                                                                          Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg           #               14051395 - #                1400                              - GAC TGG ATC AGG TAC AAC CAG TTC CGC CGC GA - #G CTG ACC CTG ACC GTG          720                                                                          Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           #              14205                                                          - CTG GAC ATC GTG AGC CTG TTC CCC AAC TAC GA - #C AGC CGC ACC TAC CCC          768                                                                          Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro           #          14350                                                              - ATC CGC ACC GTG AGC CAG CTG ACC CGC GAG AT - #T TAC ACC AAC CCC GTG          816                                                                          Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val           #      14505                                                                  - CTG GAG AAC TTC GAC GGC AGC TTC CGC GGC AG - #C GCC CAG GGC ATC GAG          864                                                                          Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu           #  14650                                                                      - GGC AGC ATC CGC AGC CCC CAC CTG ATG GAC AT - #C CTG AAC AGC ATC ACC          912                                                                          Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr           #               14851475 - #                1480                              - ATC TAC ACC GAC GCC CAC CGC GGC GAG TAC TA - #C TGG AGC GGC CAC CAG          960                                                                          Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln           #              15005                                                          - ATC ATG GCC AGC CCC GTC GGC TTC AGC GGC CC - #C GAG TTC ACC TTC CCC         1008                                                                          Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro           #          15150                                                              - CTG TAC GGC ACC ATG GGC AAC GCT GCA CCT CA - #G CAG CGC ATC GTG GCA         1056                                                                          Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala           #      15305                                                                  - CAG CTG GGC CAG GGA GTG TAC CGC ACC CTG AG - #C AGC ACC CTG TAC CGT         1104                                                                          Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg           #  15450                                                                      - CGA CCT TTC AAC ATC GGC ATC AAC AAC CAG CA - #G CTG AGC GTG CTG GAC         1152                                                                          Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp           #               15651555 - #                1560                              - GGC ACC GAG TTC GCC TAC GGC ACC AGC AGC AA - #C CTG CCC AGC GCC GTG         1200                                                                          Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           #              15805                                                          - TAC CGC AAG AGC GGC ACC GTG GAC AGC CTG GA - #C GAG ATC CCC CCT CAG         1248                                                                          Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln           #          15950                                                              - AAC AAC AAC GTG CCA CCT CGA CAG GGC TTC AG - #C CAC CGT CTG AGC CAC         1296                                                                          Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His           #      16105                                                                  - GTG AGC ATG TTC CGC AGT GGC TTC AGC AAC AG - #C AGC GTG AGC ATC ATC         1344                                                                          Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile           #  16250                                                                      - CGT GCA CCT ATG TTC AGC TGG ATT CAC CGC AG - #T GCC GAG TTC AAC AAC         1392                                                                          Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn           #               16451635 - #                1640                              - ATC ATC CCC AGC AGC CAG ATC ACC CAG ATC CC - #C CTG ACC AAG AGC ACC         1440                                                                          Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           #              16605                                                          - AAC CTG GGC AGC GGC ACC AGC GTG GTG AAG GG - #C CCC GGC TTC ACC GGC         1488                                                                          Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly           #          16750                                                              - GGC GAC ATC CTG CGC CGC ACC AGC CCC GGC CA - #G ATC AGC ACC CTG CGC         1536                                                                          Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg           #      16905                                                                  - GTG AAC ATC ACC GCC CCC CTG AGC CAG CGC TA - #C CGC GTC CGC ATC CGC         1584                                                                          Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg           #  17050                                                                      - TAC GCC AGC ACC ACC AAC CTG CAG TTC CAC AC - #C AGC ATC GAC GGC CGC         1632                                                                          Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg           #               17251715 - #                1720                              - CCC ATC AAC CAG GGC AAC TTC AGC GCC ACC AT - #G AGC AGC GGC AGC AAC         1680                                                                          Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           #              17405                                                          - CTG CAG AGC GGC AGC TTC CGC ACC GTG GGC TT - #C ACC ACC CCC TTC AAC         1728                                                                          Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn           #          17550                                                              - TTC AGC AAC GGC AGC AGC GTG TTC ACC CTG AG - #C GCC CAC GTG TTC AAC         1776                                                                          Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn           #      17705                                                                  - AGC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTG CCC GCC GAG         1824                                                                          Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu           #  17850                                                                      - GTG ACC TTC GAG GCC GAG TAC GAC CTG GAG AG - #G GCT CAG AAG GCC GTG         1872                                                                          Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val           #               18051795 - #                1800                              - AAC GAG CTG TTC ACC AGC AGC AAC CAG ATC GG - #C CTG AAG ACC GAC GTG         1920                                                                          Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val           #              18205                                                          - ACC GAC TAC CAC ATC GAT CAG GTG AGC AAC CT - #G GTG GAG TGC TTA AGC         1968                                                                          Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser           #          18350                                                              - GAC GAG TTC TGC CTG GAC GAG AAG AAG GAG CT - #G AGC GAG AAG GTG AAG         2016                                                                          Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys           #      18505                                                                  - CAC GCC AAG CGC CTG AGC GAC GAG CGC AAC CT - #G CTG CAG GAC CCC AAC         2064                                                                          His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn           #  18650                                                                      - TTC CGC GGC ATC AAC CGC CAG CTG GAC CGC GG - #C TGG CGA GGC AGC ACC         2112                                                                          Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr           #               18851875 - #                1880                              - GAT ATC ACC ATC CAG GGC GGC GAC GAC GTG TT - #C AAG GAG AAC TAC GTG         2160                                                                          Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val           #              19005                                                          - ACC CTG CTG GGC ACC TTC GAC GAG TGC TAC CC - #C ACC TAC CTG TAC CAG         2208                                                                          Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln           #          19150                                                              - AAG ATC GAC GAG AGC AAG CTG AAG GCC TAC AC - #C CGC TAC CAG CTG CGC         2256                                                                          Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg           #      19305                                                                  - GGC TAC ATC GAG GAC AGC CAG GAC CTG GAA AT - #C TAC CTG ATC CGC TAC         2304                                                                          Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr           #  19450                                                                      - AAC GCC AAG CAC GAG ACC GTG AAC GTG CCC GG - #C ACC GGC AGC CTG TGG         2352                                                                          Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp           #               19651955 - #                1960                              - CCC CTG AGC GCC CCC AGC CCC ATC GGC AAG TG - #C GGG GAG CCG AAT CGA         2400                                                                          Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg           #              19805                                                          - TGC GCT CCG CAC CTG GAG TGG AAC CCG GAC CT - #A GAC TGC AGC TGC AGG         2448                                                                          Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg           #          19950                                                              - GAC GGG GAG AAG TGC GCC CAC CAC AGC CAC CA - #C TTC AGC CTG GAC ATC         2496                                                                          Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile           #      20105                                                                  - GAC GTG GGC TGC ACC GAC CTG AAC GAG GAC CT - #G GGC GTG TGG GTG ATC         2544                                                                          Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile           #  20250                                                                      - TTC AAG ATC AAG ACC CAG GAC GGC CAC GCC CG - #C CTG GGC AAT CTA GAA         2592                                                                          Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu           #               20452035 - #                2040                              - TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GC - #A CTA GCT CGT GTG AAA         2640                                                                          Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys           #              20605                                                          - AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GA - #A AAA TTG GAA TGG GAA         2688                                                                          Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu           #          20750                                                              - ACA AAT ATT GTT TAT AAA GAG GCA AAA GAA TC - #T GTA GAT GCT TTA TTT         2736                                                                          Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe           #      20905                                                                  - GTA AAC TCT CAA TAT GAT AGA TTA CAA GCG GA - #T ACC AAC ATC GCG ATG         2784                                                                          Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met           #  21050                                                                      - ATT CAT GCG GCA GAT AAA CGC GTT CAT AGC AT - #T CGA GAA GCT TAT CTG         2832                                                                          Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu           #               21252115 - #                2120                              - CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GC - #G GCT ATT TTT GAA GAA         2880                                                                          Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu           #              21405                                                          - TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CT - #A TAT GAT GCG AGA AAT         2928                                                                          Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn           #          21550                                                              - GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TT - #A TCC TGC TGG AAC GTG         2976                                                                          Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val           #      21705                                                                  - AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AA - #C CAC CGT TCG GTC CTT         3024                                                                          Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu           #  21850                                                                      - GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CA - #A GAA GTT CGT GTC TGT         3072                                                                          Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys           #               22052195 - #                2200                              - CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GC - #G TAC AAG GAG GGA TAT         3120                                                                          Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr           #              22205                                                          - GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GA - #G AAC AAT ACA GAC GAA         3168                                                                          Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu           #          22350                                                              - CTG AAG TTT AGC AAC TGT GTA GAA GAG GAA GT - #A TAT CCA AAC AAC ACG         3216                                                                          Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr           #      22505                                                                  - GTA ACG TGT AAT GAT TAT ACT GCG ACT CAA GA - #A GAA TAT GAG GGT ACG         3264                                                                          Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr           #  22650                                                                      - TAC ACT TCT CGT AAT CGA GGA TAT GAC GGA GC - #C TAT GAA AGC AAT TCT         3312                                                                          Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser           #               22852275 - #                2280                              - TCT GTA CCA GCT GAT TAT GCA TCA GCC TAT GA - #A GAA AAA GCA TAT ACA         3360                                                                          Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr           #              23005                                                          - GAT GGA CGA AGA GAC AAT CCT TGT GAA TCT AA - #C AGA GGA TAT GGG GAT         3408                                                                          Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp           #          23150                                                              - TAC ACA CCA CTA CCA GCT GGC TAT GTG ACA AA - #A GAA TTA GAG TAC TTC         3456                                                                          Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe           #      23305                                                                  - CCA GAA ACC GAT AAG GTA TGG ATT GAG ATC GG - #A GAA ACG GAA GGA ACA         3504                                                                          Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr           #  23450                                                                      - TTC ATC GTG GAC AGC GTG GAA TTA CTT CTT AT - #G GAG GAA TAA                 #3546                                                                         Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                       2350                2355 - #                2360                              - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1181 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu         #                 15                                                          - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly         #             30                                                              - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser         #         45                                                                  - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile         #     60                                                                      - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile         # 80                                                                          - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala         #                 95                                                          - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu         #           110                                                               - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu         #       125                                                                   - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala         #   140                                                                       - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser         #               175                                                           - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg         #           190                                                               - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val         #       205                                                                   - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg         #   220                                                                       - Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro         #               255                                                           - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val         #           270                                                               - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu         #       285                                                                   - Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr         #   300                                                                       - Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro         #               335                                                           - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala         #           350                                                               - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg         #       365                                                                   - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp         #   380                                                                       - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln         #               415                                                           - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His         #           430                                                               - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile         #       445                                                                   - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn         #   460                                                                       - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly         #               495                                                           - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg         #           510                                                               - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg         #       525                                                                   - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg         #   540                                                                       - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn         #               575                                                           - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn         #           590                                                               - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu         #       605                                                                   - Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val         #   620                                                                       - Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser         #               655                                                           - Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys         #           670                                                               - His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn         #       685                                                                   - Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr         #   700                                                                       - Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln         #               735                                                           - Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg         #           750                                                               - Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr         #       765                                                                   - Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp         #   780                                                                       - Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg         #               815                                                           - Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile         #           830                                                               - Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile         #       845                                                                   - Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu         #   860                                                                       - Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu         #               895                                                           - Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe         #           910                                                               - Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met         #       925                                                                   - Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu         #   940                                                                       - Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn         #               975                                                           - Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val         #           990                                                               - Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu         #      10050                                                                  - Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys         #  10205                                                                      - Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr         #               10401030 - #                1035                              - Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu         #              10550                                                          - Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr         #          10705                                                              - Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr         #      10850                                                                  - Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser         #  11005                                                                      - Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr         #               11201110 - #                1115                              - Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp         #              11350                                                          - Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe         #          11505                                                              - Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr         #      11650                                                                  - Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                     #  11805                                                                      - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3546 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3543                                               #/product= "Full-length, hybrid,                                                             maize opt - #imized heat stable cryIA(b)"                      #"Disclosed in Figure 13 as contained in pCIB551 - #3."                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - ATG GAC AAC AAC CCC AAC ATC AAC GAG TGC AT - #C CCC TAC AAC TGC CTG           48                                                                          Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu           #          11950                                                              - AGC AAC CCC GAG GTG GAG GTG CTG GGC GGC GA - #G CGC ATC GAG ACC GGC           96                                                                          Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly           #      12105                                                                  - TAC ACC CCC ATC GAC ATC AGC CTG AGC CTG AC - #C CAG TTC CTG CTG AGC          144                                                                          Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser           #  12250                                                                      - GAG TTC GTG CCC GGC GCC GGC TTC GTG CTG GG - #C CTG GTG GAC ATC ATC          192                                                                          Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           #               12451235 - #                1240                              - TGG GGC ATC TTC GGC CCC AGC CAG TGG GAC GC - #C TTC CTG GTG CAG ATC          240                                                                          Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile           #              12605                                                          - GAG CAG CTG ATC AAC CAG CGC ATC GAG GAG TT - #C GCC CGC AAC CAG GCC          288                                                                          Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala           #          12750                                                              - ATC AGC CGC CTG GAG GGC CTG AGC AAC CTG TA - #C CAA ATC TAC GCC GAG          336                                                                          Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu           #      12905                                                                  - AGC TTC CGC GAG TGG GAG GCC GAC CCC ACC AA - #C CCC GCC CTG CGC GAG          384                                                                          Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu           #  13050                                                                      - GAG ATG CGC ATC CAG TTC AAC GAC ATG AAC AG - #C GCC CTG ACC ACC GCC          432                                                                          Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala           #               13251315 - #                1320                              - ATC CCC CTG TTC GCC GTG CAG AAC TAC CAG GT - #G CCC CTG CTG AGC GTG          480                                                                          Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           #              13405                                                          - TAC GTG CAG GCC GCC AAC CTG CAC CTG AGC GT - #G CTG CGC GAC GTC AGC          528                                                                          Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser           #          13550                                                              - GTG TTC GGC CAG CGC TGG GGC TTC GAC GCC GC - #C ACC ATC AAC AGC CGC          576                                                                          Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg           #      13705                                                                  - TAC AAC GAC CTG ACC CGC CTG ATC GGC AAC TA - #C ACC GAC CAC GCC GTG          624                                                                          Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val           #  13850                                                                      - CGC TGG TAC AAC ACC GGC CTG GAG CGC GTG TG - #G GGT CCC GAC AGC CGC          672                                                                          Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg           #               14051395 - #                1400                              - GAC TGG ATC AGG TAC AAC CAG TTC CGC CGC GA - #G CTG ACC CTG ACC GTG          720                                                                          Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           #              14205                                                          - CTG GAC ATC GTG AGC CTG TTC CCC AAC TAC GA - #C AGC CGC ACC TAC CCC          768                                                                          Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro           #          14350                                                              - ATC CGC ACC GTG AGC CAG CTG ACC CGC GAG AT - #T TAC ACC AAC CCC GTG          816                                                                          Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val           #      14505                                                                  - CTG GAG AAC TTC GAC GGC AGC TTC CGC GGC AG - #C GCC CAG GGC ATC GAG          864                                                                          Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu           #  14650                                                                      - GGC AGC ATC CGC AGC CCC CAC CTG ATG GAC AT - #C CTG AAC AGC ATC ACC          912                                                                          Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr           #               14851475 - #                1480                              - ATC TAC ACC GAC GCC CAC CGC GGC GAG TAC TA - #C TGG AGC GGC CAC CAG          960                                                                          Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln           #              15005                                                          - ATC ATG GCC AGC CCC GTC GGC TTC AGC GGC CC - #C GAG TTC ACC TTC CCC         1008                                                                          Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro           #          15150                                                              - CTG TAC GGC ACC ATG GGC AAC GCT GCA CCT CA - #G CAG CGC ATC GTG GCA         1056                                                                          Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala           #      15305                                                                  - CAG CTG GGC CAG GGA GTG TAC CGC ACC CTG AG - #C AGC ACC CTG TAC CGT         1104                                                                          Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg           #  15450                                                                      - CGA CCT TTC AAC ATC GGC ATC AAC AAC CAG CA - #G CTG AGC GTG CTG GAC         1152                                                                          Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp           #               15651555 - #                1560                              - GGC ACC GAG TTC GCC TAC GGC ACC AGC AGC AA - #C CTG CCC AGC GCC GTG         1200                                                                          Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           #              15805                                                          - TAC CGC AAG AGC GGC ACC GTG GAC AGC CTG GA - #C GAG ATC CCC CCT CAG         1248                                                                          Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln           #          15950                                                              - AAC AAC AAC GTG CCA CCT CGA CAG GGC TTC AG - #C CAC CGT CTG AGC CAC         1296                                                                          Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His           #      16105                                                                  - GTG AGC ATG TTC CGC AGT GGC TTC AGC AAC AG - #C AGC GTG AGC ATC ATC         1344                                                                          Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile           #  16250                                                                      - CGT GCA CCT ATG TTC AGC TGG ATT CAC CGC AG - #T GCC GAG TTC AAC AAC         1392                                                                          Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn           #               16451635 - #                1640                              - ATC ATC CCC AGC AGC CAG ATC ACC CAG ATC CC - #C CTG ACC AAG AGC ACC         1440                                                                          Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           #              16605                                                          - AAC CTG GGC AGC GGC ACC AGC GTG GTG AAG GG - #C CCC GGC TTC ACC GGC         1488                                                                          Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly           #          16750                                                              - GGC GAC ATC CTG CGC CGC ACC AGC CCC GGC CA - #G ATC AGC ACC CTG CGC         1536                                                                          Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg           #      16905                                                                  - GTG AAC ATC ACC GCC CCC CTG AGC CAG CGC TA - #C CGC GTC CGC ATC CGC         1584                                                                          Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg           #  17050                                                                      - TAC GCC AGC ACC ACC AAC CTG CAG TTC CAC AC - #C AGC ATC GAC GGC CGC         1632                                                                          Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg           #               17251715 - #                1720                              - CCC ATC AAC CAG GGC AAC TTC AGC GCC ACC AT - #G AGC AGC GGC AGC AAC         1680                                                                          Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           #              17405                                                          - CTG CAG AGC GGC AGC TTC CGC ACC GTG GGC TT - #C ACC ACC CCC TTC AAC         1728                                                                          Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn           #          17550                                                              - TTC AGC AAC GGC AGC AGC GTG TTC ACC CTG AG - #C GCC CAC GTG TTC AAC         1776                                                                          Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn           #      17705                                                                  - AGC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTG CCC GCC GAG         1824                                                                          Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu           #  17850                                                                      - GTG ACC TTC GAG GCC GAG TAC GAC CTG GAG AG - #G GCT CAG AAG GCC GTG         1872                                                                          Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val           #               18051795 - #                1800                              - AAC GAG CTG TTC ACC AGC AGC AAC CAG ATC GG - #C CTG AAG ACC GAC GTG         1920                                                                          Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val           #              18205                                                          - ACC GAC TAC CAC ATC GAC CAG GTG AGC AAC CT - #G GTG GAG TGC TTA AGC         1968                                                                          Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser           #          18350                                                              - GAC GAG TTC TGC CTG GAC GAG AAG AAG GAG CT - #G AGC GAG AAG GTG AAG         2016                                                                          Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys           #      18505                                                                  - CAC GCC AAG CGC CTG AGC GAC GAG CGC AAC CT - #G CTG CAG GAC CCC AAC         2064                                                                          His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn           #  18650                                                                      - TTC CGC GGC ATC AAC CGC CAG CTG GAC CGC GG - #C TGG CGA GGC AGC ACC         2112                                                                          Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr           #               18851875 - #                1880                              - GAT ATC ACC ATC CAG GGC GGC GAC GAC GTG TT - #C AAG GAG AAC TAC GTG         2160                                                                          Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val           #              19005                                                          - ACC CTG CAG GGC ACC TTC GAC GAG TGC TAC CC - #C ACC TAC CTG TAC CAG         2208                                                                          Thr Leu Gln Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln           #          19150                                                              - CCG ATC GAC GAG AGC AAG CTG AAG GCC TAC AC - #C CGC TAC CAG CTG CGC         2256                                                                          Pro Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg           #      19305                                                                  - GGC TAC ATC GAG GAC AGC CAG GAC CTG GAA AT - #C TAC CTG ATC CGC TAC         2304                                                                          Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr           #  19450                                                                      - AAC GCC AAG CAC GAG ACC GTG AAC GTG CCC GG - #C ACC GGC AGC CTG TGG         2352                                                                          Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp           #               19651955 - #                1960                              - CCC CTG AGC GCC CCC AGC CCC ATC GGC AAG TG - #C GGG GAG CCG AAT CGA         2400                                                                          Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg           #              19805                                                          - TGC GCT CCG CAC CTG GAG TGG AAC CCG GAC CT - #A GAC TGC AGC TGC AGG         2448                                                                          Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg           #          19950                                                              - GAC GGG GAG AAG TGC GCC CAC CAC AGC CAC CA - #C TTC AGC CTG GAC ATC         2496                                                                          Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile           #      20105                                                                  - GAC GTG GGC TGC ACC GAC CTG AAC GAG GAC CT - #G GGC GTG TGG GTG ATC         2544                                                                          Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile           #  20250                                                                      - TTC AAG ATC AAG ACC CAG GAC GGC CAC GCC CG - #C CTG GGC AAT CTA GAG         2592                                                                          Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu           #               20452035 - #                2040                              - TTC CTG GAG GAG AAG CCC CTG GTG GGC GAG GC - #C CTG GCC CGC GTG AAG         2640                                                                          Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys           #              20605                                                          - CGC GCC GAG AAG AAG TGG CGC GAC AAG CGC GA - #G AAG CTG GAG TGG GAG         2688                                                                          Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu           #          20750                                                              - ACC AAC ATC GTG TAC AAG GAG GCC AAG GAG AG - #C GTG GAC GCC CTG TTC         2736                                                                          Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe           #      20905                                                                  - GTG AAC AGC CAG TAC GAC CGC CTG CAG GCC GA - #C ACC AAC ATC GCC ATG         2784                                                                          Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met           #  21050                                                                      - ATC CAC GCC GCC GAC AAG CGC GTG CAC AGC AT - #T CGC GAG GCC TAC CTG         2832                                                                          Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu           #               21252115 - #                2120                              - CCC GAG CTG AGC GTG ATC CCC GGC GTG AAC GC - #C GCC ATC TTC GAG GAA         2880                                                                          Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu           #              21405                                                          - CTC GAG GGC CGC ATC TTC ACC GCC TTC AGC CT - #G TAC GAC GCC CGC AAC         2928                                                                          Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn           #          21550                                                              - GTG ATC AAG AAC GGC GAC TTC AAC AAC GGC CT - #G AGC TGC TGG AAC GTG         2976                                                                          Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val           #      21705                                                                  - AAG GGC CAC GTG GAC GTG GAG GAG CAG AAC AA - #C CAC CGC AGC GTG CTG         3024                                                                          Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu           #  21850                                                                      - GTG GTG CCC GAG TGG GAG GCC GAG GTG AGC CA - #G GAG GTG CGC GTG TGC         3072                                                                          Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys           #               22052195 - #                2200                              - CCC GGC CGC GGC TAC ATC CTG CGC GTG ACC GC - #C TAC AAG GAG GGC TAC         3120                                                                          Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr           #              22205                                                          - GGC GAG GGC TGC GTG ACC ATC CAC GAG ATC GA - #G AAC AAC ACC GAC GAG         3168                                                                          Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu           #          22350                                                              - CTC AAG TTC AGC AAC TGC GTG GAG GAG GAG GT - #T TAC CCC AAC AAC ACC         3216                                                                          Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr           #      22505                                                                  - GTG ACC TGC AAC GAC TAC ACC GCG ACC CAG GA - #G GAG TAC GAA GGC ACC         3264                                                                          Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr           #  22650                                                                      - TAC ACC TCT CGC AAC AGG GGT TAC GAC GGC GC - #C TAC GAG TCC AAC AGC         3312                                                                          Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser           #               22852275 - #                2280                              - TCC GTG CCA GCC GAC TAC GCC AGC GCC TAC GA - #G GAG AAA GCC TAC ACC         3360                                                                          Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr           #              23005                                                          - GAC GGT AGA CGC GAC AAC CCA TGT GAG AGC AA - #C AGA GGC TAC GGC GAC         3408                                                                          Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp           #          23150                                                              - TAC ACC CCC CTG CCC GCT GGA TAC GTG ACC AA - #G GAG CTG GAG TAC TTC         3456                                                                          Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe           #      23305                                                                  - CCC GAG ACC GAC AAG GTG TGG ATC GAG ATT GG - #C GAG ACC GAG GGC ACC         3504                                                                          Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr           #  23450                                                                      - TTC ATC GTG GAC AGC GTG GAG CTG CTG CTG AT - #G GAG GAG TAG                 #3546                                                                         Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                       2350                2355 - #                2360                              - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1181 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu         #                 15                                                          - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly         #             30                                                              - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser         #         45                                                                  - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile         #     60                                                                      - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile         # 80                                                                          - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala         #                 95                                                          - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu         #           110                                                               - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu         #       125                                                                   - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala         #   140                                                                       - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser         #               175                                                           - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg         #           190                                                               - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val         #       205                                                                   - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg         #   220                                                                       - Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro         #               255                                                           - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val         #           270                                                               - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu         #       285                                                                   - Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr         #   300                                                                       - Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro         #               335                                                           - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala         #           350                                                               - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg         #       365                                                                   - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp         #   380                                                                       - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln         #               415                                                           - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His         #           430                                                               - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile         #       445                                                                   - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn         #   460                                                                       - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly         #               495                                                           - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg         #           510                                                               - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg         #       525                                                                   - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg         #   540                                                                       - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn         #               575                                                           - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn         #           590                                                               - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu         #       605                                                                   - Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val         #   620                                                                       - Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser         #               655                                                           - Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys         #           670                                                               - His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn         #       685                                                                   - Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr         #   700                                                                       - Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Leu Gln Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln         #               735                                                           - Pro Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg         #           750                                                               - Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr         #       765                                                                   - Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp         #   780                                                                       - Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg         #               815                                                           - Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile         #           830                                                               - Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile         #       845                                                                   - Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu         #   860                                                                       - Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu         #               895                                                           - Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe         #           910                                                               - Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met         #       925                                                                   - Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu         #   940                                                                       - Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn         #               975                                                           - Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val         #           990                                                               - Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu         #      10050                                                                  - Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys         #  10205                                                                      - Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr         #               10401030 - #                1035                              - Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu         #              10550                                                          - Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr         #          10705                                                              - Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr         #      10850                                                                  - Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser         #  11005                                                                      - Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr         #               11201110 - #                1115                              - Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp         #              11350                                                          - Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe         #          11505                                                              - Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr         #      11650                                                                  - Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                     #  11805                                                                      - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3547 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3543                                               #/product= "Full-length, hybrid,                                                             maize opt - #imized heat stable cryIA(b)"                      #"Disclosed in Figure 15 as contained in pCIB551 - #4."                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - ATG GAC AAC AAC CCC AAC ATC AAC GAG TGC AT - #C CCC TAC AAC TGC CTG           48                                                                          Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu           #          11950                                                              - AGC AAC CCC GAG GTG GAG GTG CTG GGC GGC GA - #G CGC ATC GAG ACC GGC           96                                                                          Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly           #      12105                                                                  - TAC ACC CCC ATC GAC ATC AGC CTG AGC CTG AC - #C CAG TTC CTG CTG AGC          144                                                                          Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser           #  12250                                                                      - GAG TTC GTG CCC GGC GCC GGC TTC GTG CTG GG - #C CTG GTG GAC ATC ATC          192                                                                          Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           #               12451235 - #                1240                              - TGG GGC ATC TTC GGC CCC AGC CAG TGG GAC GC - #C TTC CTG GTG CAG ATC          240                                                                          Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile           #              12605                                                          - GAG CAG CTG ATC AAC CAG CGC ATC GAG GAG TT - #C GCC CGC AAC CAG GCC          288                                                                          Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala           #          12750                                                              - ATC AGC CGC CTG GAG GGC CTG AGC AAC CTG TA - #C CAA ATC TAC GCC GAG          336                                                                          Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu           #      12905                                                                  - AGC TTC CGC GAG TGG GAG GCC GAC CCC ACC AA - #C CCC GCC CTG CGC GAG          384                                                                          Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu           #  13050                                                                      - GAG ATG CGC ATC CAG TTC AAC GAC ATG AAC AG - #C GCC CTG ACC ACC GCC          432                                                                          Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala           #               13251315 - #                1320                              - ATC CCC CTG TTC GCC GTG CAG AAC TAC CAG GT - #G CCC CTG CTG AGC GTG          480                                                                          Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           #              13405                                                          - TAC GTG CAG GCC GCC AAC CTG CAC CTG AGC GT - #G CTG CGC GAC GTC AGC          528                                                                          Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser           #          13550                                                              - GTG TTC GGC CAG CGC TGG GGC TTC GAC GCC GC - #C ACC ATC AAC AGC CGC          576                                                                          Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg           #      13705                                                                  - TAC AAC GAC CTG ACC CGC CTG ATC GGC AAC TA - #C ACC GAC CAC GCC GTG          624                                                                          Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val           #  13850                                                                      - CGC TGG TAC AAC ACC GGC CTG GAG CGC GTG TG - #G GGT CCC GAC AGC CGC          672                                                                          Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg           #               14051395 - #                1400                              - GAC TGG ATC AGG TAC AAC CAG TTC CGC CGC GA - #G CTG ACC CTG ACC GTG          720                                                                          Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           #              14205                                                          - CTG GAC ATC GTG AGC CTG TTC CCC AAC TAC GA - #C AGC CGC ACC TAC CCC          768                                                                          Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro           #          14350                                                              - ATC CGC ACC GTG AGC CAG CTG ACC CGC GAG AT - #T TAC ACC AAC CCC GTG          816                                                                          Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val           #      14505                                                                  - CTG GAG AAC TTC GAC GGC AGC TTC CGC GGC AG - #C GCC CAG GGC ATC GAG          864                                                                          Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu           #  14650                                                                      - GGC AGC ATC CGC AGC CCC CAC CTG ATG GAC AT - #C CTG AAC AGC ATC ACC          912                                                                          Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr           #               14851475 - #                1480                              - ATC TAC ACC GAC GCC CAC CGC GGC GAG TAC TA - #C TGG AGC GGC CAC CAG          960                                                                          Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln           #              15005                                                          - ATC ATG GCC AGC CCC GTC GGC TTC AGC GGC CC - #C GAG TTC ACC TTC CCC         1008                                                                          Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro           #          15150                                                              - CTG TAC GGC ACC ATG GGC AAC GCT GCA CCT CA - #G CAG CGC ATC GTG GCA         1056                                                                          Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala           #      15305                                                                  - CAG CTG GGC CAG GGA GTG TAC CGC ACC CTG AG - #C AGC ACC CTG TAC CGT         1104                                                                          Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg           #  15450                                                                      - CGA CCT TTC AAC ATC GGC ATC AAC AAC CAG CA - #G CTG AGC GTG CTG GAC         1152                                                                          Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp           #               15651555 - #                1560                              - GGC ACC GAG TTC GCC TAC GGC ACC AGC AGC AA - #C CTG CCC AGC GCC GTG         1200                                                                          Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           #              15805                                                          - TAC CGC AAG AGC GGC ACC GTG GAC AGC CTG GA - #C GAG ATC CCC CCT CAG         1248                                                                          Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln           #          15950                                                              - AAC AAC AAC GTG CCA CCT CGA CAG GGC TTC AG - #C CAC CGT CTG AGC CAC         1296                                                                          Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His           #      16105                                                                  - GTG AGC ATG TTC CGC AGT GGC TTC AGC AAC AG - #C AGC GTG AGC ATC ATC         1344                                                                          Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile           #  16250                                                                      - CGT GCA CCT ATG TTC AGC TGG ATT CAC CGC AG - #T GCC GAG TTC AAC AAC         1392                                                                          Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn           #               16451635 - #                1640                              - ATC ATC CCC AGC AGC CAG ATC ACC CAG ATC CC - #C CTG ACC AAG AGC ACC         1440                                                                          Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           #              16605                                                          - AAC CTG GGC AGC GGC ACC AGC GTG GTG AAG GG - #C CCC GGC TTC ACC GGC         1488                                                                          Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly           #          16750                                                              - GGC GAC ATC CTG CGC CGC ACC AGC CCC GGC CA - #G ATC AGC ACC CTG CGC         1536                                                                          Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg           #      16905                                                                  - GTG AAC ATC ACC GCC CCC CTG AGC CAG CGC TA - #C CGC GTC CGC ATC CGC         1584                                                                          Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg           #  17050                                                                      - TAC GCC AGC ACC ACC AAC CTG CAG TTC CAC AC - #C AGC ATC GAC GGC CGC         1632                                                                          Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg           #               17251715 - #                1720                              - CCC ATC AAC CAG GGC AAC TTC AGC GCC ACC AT - #G AGC AGC GGC AGC AAC         1680                                                                          Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           #              17405                                                          - CTG CAG AGC GGC AGC TTC CGC ACC GTG GGC TT - #C ACC ACC CCC TTC AAC         1728                                                                          Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn           #          17550                                                              - TTC AGC AAC GGC AGC AGC GTG TTC ACC CTG AG - #C GCC CAC GTG TTC AAC         1776                                                                          Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn           #      17705                                                                  - AGC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTG CCC GCC GAG         1824                                                                          Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu           #  17850                                                                      - GTG ACC TTC GAG GCC GAG TAC GAC CTG GAG AG - #G GCT CAG AAG GCC GTG         1872                                                                          Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val           #               18051795 - #                1800                              - AAC GAG CTG TTC ACC AGC AGC AAC CAG ATC GG - #C CTG AAG ACC GAC GTG         1920                                                                          Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val           #              18205                                                          - ACC GAC TAC CAC ATC GAT CAA GTA TCC AAT TT - #A GTT GAG TGT TTA TCT         1968                                                                          Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser           #          18350                                                              - GAT GAA TTT TGT CTG GAT GAA AAA AAA GAA TT - #G TCC GAG AAA GTC AAA         2016                                                                          Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys           #      18505                                                                  - CAT GCG AAG CGA CTT AGT GAT GAG CGG AAT TT - #A CTT CAA GAT CCA AAC         2064                                                                          His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn           #  18650                                                                      - TTT AGA GGG ATC AAT AGA CAA CTA GAC CGT GG - #C TGG AGA GGA AGT ACG         2112                                                                          Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr           #               18851875 - #                1880                              - GAT ATT ACC ATC CAA GGA GGC GAT GAC GTA TT - #C AAA GAG AAT TAC GTT         2160                                                                          Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val           #              19005                                                          - ACG CTA TTG GGT ACC TTT GAT GAG TGC TAT CC - #A ACG TAT TTA TAT CAA         2208                                                                          Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln           #          19150                                                              - AAA ATA GAT GAG TCG AAA TTA AAA GCC TAT AC - #C CGT TAC CAA TTA AGA         2256                                                                          Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg           #      19305                                                                  - GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA AT - #C TAT TTA ATT CGC TAC         2304                                                                          Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr           #  19450                                                                      - AAT GCC AAA CAC GAA ACA GTA AAT GTG CCA GG - #T ACG GGT TCC TTA TGG         2352                                                                          Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp           #               19651955 - #                1960                              - CCG CTT TCA GCC CCA AGT CCA ATC GGC AAG TG - #C GGG GAG CCG AAT CGA         2400                                                                          Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg           #              19805                                                          - TGC GCT CCG CAC CTG GAG TGG AAC CCG GAC CT - #A GAC TGC AGC TGC AGG         2448                                                                          Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg           #          19950                                                              - GAC GGG GAG AAG TGC GCC CAC CAC AGC CAC CA - #C TTC AGC CTG GAC ATC         2496                                                                          Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile           #      20105                                                                  - GAC GTG GGC TGC ACC GAC CTG AAC GAG GAC CT - #G GGC GTG TGG GTG ATC         2544                                                                          Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile           #  20250                                                                      - TTC AAG ATC AAG ACC CAG GAC GGC CAC GCC CG - #C CTG GGC AAT CTA GAA         2592                                                                          Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu           #               20452035 - #                2040                              - TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GC - #A CTA GCT CGT GTG AAA         2640                                                                          Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys           #              20605                                                          - AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GA - #A AAA TTG GAA TGG GAA         2688                                                                          Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu           #          20750                                                              - ACA AAT ATT GTT TAT AAA GAG GCA AAA GAA TC - #T GTA GAT GCT TTA TTT         2736                                                                          Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe           #      20905                                                                  - GTA AAC TCT CAA TAT GAT AGA TTA CAA GCG GA - #T ACC AAC ATC GCG ATG         2784                                                                          Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met           #  21050                                                                      - ATT CAT GCG GCA GAT AAA CGC GTT CAT AGC AT - #T CGA GAA GCT TAT CTG         2832                                                                          Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu           #               21252115 - #                2120                              - CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GC - #G GCT ATT TTT GAA GAA         2880                                                                          Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu           #              21405                                                          - TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CT - #A TAT GAT GCG AGA AAT         2928                                                                          Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn           #          21550                                                              - GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TT - #A TCC TGC TGG AAC GTG         2976                                                                          Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val           #      21705                                                                  - AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AA - #C CAC CGT TCG GTC CTT         3024                                                                          Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu           #  21850                                                                      - GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CA - #A GAA GTT CGT GTC TGT         3072                                                                          Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys           #               22052195 - #                2200                              - CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GC - #G TAC AAG GAG GGA TAT         3120                                                                          Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr           #              22205                                                          - GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GA - #G AAC AAT ACA GAC GAA         3168                                                                          Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu           #          22350                                                              - CTG AAG TTT AGC AAC TGT GTA GAA GAG GAA GT - #A TAT CCA AAC AAC ACG         3216                                                                          Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr           #      22505                                                                  - GTA ACG TGT AAT GAT TAT ACT GCG ACT CAA GA - #A GAA TAT GAG GGT ACG         3264                                                                          Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr           #  22650                                                                      - TAC ACT TCT CGT AAT CGA GGA TAT GAC GGA GC - #C TAT GAA AGC AAT TCT         3312                                                                          Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser           #               22852275 - #                2280                              - TCT GTA CCA GCT GAT TAT GCA TCA GCC TAT GA - #A GAA AAA GCA TAT ACA         3360                                                                          Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr           #              23005                                                          - GAT GGA CGA AGA GAC AAT CCT TGT GAA TCT AA - #C AGA GGA TAT GGG GAT         3408                                                                          Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp           #          23150                                                              - TAC ACA CCA CTA CCA GCT GGC TAT GTG ACA AA - #A GAA TTA GAG TAC TTC         3456                                                                          Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe           #      23305                                                                  - CCA GAA ACC GAT AAG GTA TGG ATT GAG ATC GG - #A GAA ACG GAA GGA ACA         3504                                                                          Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr           #  23450                                                                      - TTC ATC GTG GAC AGC GTG GAA TTA CTT CTT AT - #G GAG GAA TAAG                #3547                                                                         Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                       2350                2355 - #                2360                              - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1181 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu         #                 15                                                          - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly         #             30                                                              - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser         #         45                                                                  - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile         #     60                                                                      - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile         # 80                                                                          - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala         #                 95                                                          - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu         #           110                                                               - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu         #       125                                                                   - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala         #   140                                                                       - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser         #               175                                                           - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg         #           190                                                               - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val         #       205                                                                   - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg         #   220                                                                       - Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro         #               255                                                           - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val         #           270                                                               - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu         #       285                                                                   - Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr         #   300                                                                       - Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro         #               335                                                           - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala         #           350                                                               - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg         #       365                                                                   - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp         #   380                                                                       - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln         #               415                                                           - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His         #           430                                                               - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile         #       445                                                                   - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn         #   460                                                                       - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly         #               495                                                           - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg         #           510                                                               - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg         #       525                                                                   - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg         #   540                                                                       - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn         #               575                                                           - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn         #           590                                                               - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu         #       605                                                                   - Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val         #   620                                                                       - Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser         #               655                                                           - Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys         #           670                                                               - His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn         #       685                                                                   - Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr         #   700                                                                       - Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln         #               735                                                           - Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg         #           750                                                               - Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr         #       765                                                                   - Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp         #   780                                                                       - Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg         #               815                                                           - Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile         #           830                                                               - Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile         #       845                                                                   - Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu         #   860                                                                       - Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu         #               895                                                           - Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe         #           910                                                               - Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met         #       925                                                                   - Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu         #   940                                                                       - Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn         #               975                                                           - Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val         #           990                                                               - Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu         #      10050                                                                  - Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys         #  10205                                                                      - Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr         #               10401030 - #                1035                              - Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu         #              10550                                                          - Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr         #          10705                                                              - Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr         #      10850                                                                  - Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser         #  11005                                                                      - Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr         #               11201110 - #                1115                              - Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp         #              11350                                                          - Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe         #          11505                                                              - Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr         #      11650                                                                  - Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                     #  11805                                                                      - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 4817 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: join(1839..2 - #141, 2239..2547, 2641..2718,          2794                                                                                         ..2871, 3 - #001..3135, 3236..3370)                            #/product= "maize TrpA"ORMATION:                                              #"Maize TrpA sequence as disclosed in Figure 24. - #"                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: TATA.sub.-- - #signal                                           (B) LOCATION: 1594..1599                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: CAAT.sub.-- - #signal                                           (B) LOCATION: 1495..1499                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: promoter                                                        (B) LOCATION: 39..1838                                              #/function= "Promoter sequence used                                                          in pCIB44 - #33"                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - GAATTCGGAT CCATTAAAGA AGTCTTTGAA CAGATTCTAG AGATCTAGTT TA - #ATGAGCTC         60                                                                          - CCAAAAGTCT TGAAAAAATT CAGCGGGGAG GCCATTAGGG CAGGGGTACT GT - #TATGTTTT        120                                                                          - AAAGAGAACA CCACTTTCTT GATCTCTTCT AAAGAGAAAT GTTTTGTAAG AA - #GGATCCTG        180                                                                          - TCCTCCTCAT CCAACCTTTT CATCGGCAAA TTTTTCATAG AGATATTAGA GG - #CAAGAGAG        240                                                                          - GGGCCAAAAA GATCCATGTA AATGGAAGTG GCCACCTGGT TGATACCTCC CT - #CATCTTCA        300                                                                          - ACAGAAAATC CATTATGAAA AAGTGAATGG ATTTTAAACT CTTCTTTTTC TT - #CCCTTTTG        360                                                                          - CAATGAGCTG AAAATATCTG GTATTATTCT CATCACCCTC ATTAATGAAT CT - #GTCCCTAG        420                                                                          - CAATTTGCTT TCTCTTGATC CCTTCTGCAG CCACCATGTT TCTTAAATTC CA - #CTCCATAT        480                                                                          - CAAGCTTTTC CAATCTATCA GAATCTGAGA TGGCTGCAAT CTCTCTCATT TT - #CTCAAGGA        540                                                                          - TATCGATGTT ATCCATAAGG TATTTCTTGA ACTTCTTATA TTTCCCTTCG AC - #ATTTATAT        600                                                                          - TCCATCCTTT CAACATTTTT TTGTTCAATC TTTTTTGTTT TTTTCCTTTC CA - #AACATCGA        660                                                                          - TACATTTCCT GCTCCTCACA GGTAAGGACG AGCTTTCAAA AAACCTTCTG CT - #TTAAAGTC        720                                                                          - AGGTCTGAGC CTCCAGCAAA GCTCACATAT CTAAAGTCCC TCTTCTTAGT TG - #GGACAGAG        780                                                                          - TCAGTGCTAA GACACATGGG AACATGACCA GAAAAAAAAA ATCATATTTA GC - #CCAGAGAC        840                                                                          - AACAATATTC TTGTACTGCA AGTCTCGTTA TGGGCTAGCA AAGGAATCTA CC - #CAACTTCT        900                                                                          - CAAATGTGTT GGGATGTCAA GTATATAGAC TATTCATCAG TTCCAACTCT AT - #CAAACTGT        960                                                                          - GCAGCTCAAT TATAGAGTTG AATAAAGTGC TCCATCTATT TGTTCTTATC CT - #CATATTTG       1020                                                                          - GTTAAGATAT TAAAATCACC TCCCACCAAC ATTTAAAGTG CACCATTTAA AG - #TGGCTCGC       1080                                                                          - GAGCACCAAA CCGCTGAAAA CCGGAAATGT TTAGCACGTT GGCAGCGGGA CC - #CTTTTCTA       1140                                                                          - TCTCATCGTG TTCTTCGTTG TCCACCACGG CCCACGGGCC AACGCTCCTC CA - #TCCTGTAG       1200                                                                          - TGTAGAGTAT ATTCCATTTG CGACCGAGCC GAGCATCGAT CCAGCCACAC TG - #GCCACTGC       1260                                                                          - CAGCCAGCCA TGTGGCACTC CTACGTATAC TACGTGAGGT GAGATTCACT CA - #CATGGGAT       1320                                                                          - GGGACCGAGA TATTTTACTG CTGTGGTTGT GTGAGAGATA ATAAAGCATT TA - #TGACGATT       1380                                                                          - GCTGAACAGC ACACACCATG CGTCCAGATA GAGAAAGCTT TCTCTCTTTA TT - #CGCATGCA       1440                                                                          - TGTTTCATTA TCTTTTATCA TATATATATA ACACATATTA AATGATTCTT CG - #TTCCAATT       1500                                                                          - TATAATTCAT TTGACTTTTT TATCCACCGA TGCTCGTTTT ATTAAAAAAA AT - #ATTATAAT       1560                                                                          - TATTGTTACT TTTTGTTGTA ATATTGTTTA GCATATAATA AACTTTGATA CT - #AGTATGTT       1620                                                                          - TCCGAGCAAA AAAAAATATT AATATTTAGA TTACGAGCCC ATTAATTAAT TA - #TATTCGAG       1680                                                                          - ACAAGCGAAG CAAAGCAAAG CAAGCTAATG TTGCCCCTGC TGTGCATGCA GA - #GGCCCGCT       1740                                                                          - CTTGCTATAA ACGAGGCAGC TAGACGCGAC TCGACTCATC AGCCTCATCA AC - #CTCGACGA       1800                                                                          #GCG CCC       1853CAGG TTGTTGCACA GAAGCGAC ATG GCT TTC                       #      Met Ala Phe Ala Pro                                                    #     5  1                                                                    - AAA ACG TCC TCC TCC TCC TCG CTG TCC TCG GC - #G TTG CAG GCA GCT CAG         1901                                                                          Lys Thr Ser Ser Ser Ser Ser Leu Ser Ser Al - #a Leu Gln Ala Ala Gln           #                 20                                                          - TCG CCG CCG CTG CTC CTG AGG CGG ATG TCG TC - #G ACC GCA ACA CCG AGA         1949                                                                          Ser Pro Pro Leu Leu Leu Arg Arg Met Ser Se - #r Thr Ala Thr Pro Arg           #             35                                                              - CGG AGG TAC GAC GCG GCC GTC GTC GTC ACT AC - #C ACC ACC ACT GCT AGA         1997                                                                          Arg Arg Tyr Asp Ala Ala Val Val Val Thr Th - #r Thr Thr Thr Ala Arg           #         50                                                                  - GCT GCG GCG GCT GCT GTC ACG GTT CCC GCC GC - #C CCG CCG CAG GCG GGC         2045                                                                          Ala Ala Ala Ala Ala Val Thr Val Pro Ala Al - #a Pro Pro Gln Ala Gly           #     65                                                                      - CGC CGC CGC CGG TGC CAC CAA AGC AAG CGG CG - #G CAC CCG CAG AGG AGG         2093                                                                          Arg Arg Arg Arg Cys His Gln Ser Lys Arg Ar - #g His Pro Gln Arg Arg           # 85                                                                          - AGC CGT CCG GTG TCG GAC ACC ATG GCG GCG CT - #C ATG GCC AAG GGC AAG         2141                                                                          Ser Arg Pro Val Ser Asp Thr Met Ala Ala Le - #u Met Ala Lys Gly Lys           #                100                                                          - GTTCGTATAG TACGCGCGCG TGTCGTCGTC GTTATTTTGC GCATAGGCGC GG - #ACATACAC       2201                                                                          #ATC CCG TAC    2256CAG CTAGATCATC GGTGCAG ACG GCG TTC                        #     Thr Ala Phe Ile Pro Tyr                                                 #                 105                                                         - ATC ACC GCC GGC GAC CCG GAC CTA GCG ACG AC - #G GCC GAG GCG CTG CGT         2304                                                                          Ile Thr Ala Gly Asp Pro Asp Leu Ala Thr Th - #r Ala Glu Ala Leu Arg           #       120                                                                   - CTG CTG GAC GGC TGT GGC GCC GAC GTC ATC GA - #G CTG GGG GTA CCC TGC         2352                                                                          Leu Leu Asp Gly Cys Gly Ala Asp Val Ile Gl - #u Leu Gly Val Pro Cys           #   135                                                                       - TCG GAC CCC TAC ATC GAC GGG CCC ATC ATC CA - #G GCG TCG GTG GCG CGG         2400                                                                          Ser Asp Pro Tyr Ile Asp Gly Pro Ile Ile Gl - #n Ala Ser Val Ala Arg           140                 1 - #45                 1 - #50                 1 -       #55                                                                           - GCT CTG GCC AGC GGC ACC ACC ATG GAC GCC GT - #G CTG GAG ATG CTG AGG         2448                                                                          Ala Leu Ala Ser Gly Thr Thr Met Asp Ala Va - #l Leu Glu Met Leu Arg           #               170                                                           - GAG GTG ACG CCG GAG CTG TCG TGC CCC GTG GT - #G CTC CTC TCC TAC TAC         2496                                                                          Glu Val Thr Pro Glu Leu Ser Cys Pro Val Va - #l Leu Leu Ser Tyr Tyr           #           185                                                               - AAG CCC ATC ATG TCT CGC AGC TTG GCC GAG AT - #G AAA GAG GCG GGG GTC         2544                                                                          Lys Pro Ile Met Ser Arg Ser Leu Ala Glu Me - #t Lys Glu Ala Gly Val           #       200                                                                   - CAC GGTAACTATA GCTAGCTCTT CCGATCCCCC TTCAATTAAT TAATTTATA - #G              2597                                                                          His                                                                           - TAGTCCATTC ATGTGATGAT TTTTGTTTTT CTTTTTACTG ACA GGT CT - #T ATA GTG         2652                                                                          #            Gly Leu Ile Va - #l                                              #            205                                                              - CCT GAT CTC CCG TAC GTG GCC GCG CAC TCG CT - #G TGG AGT GAA GCC AAG         2700                                                                          Pro Asp Leu Pro Tyr Val Ala Ala His Ser Le - #u Trp Ser Glu Ala Lys           #   220                                                                       - AAC AAC AAC CTG GAG CTG GTAGGTTGAA TTAAGTTGAT GC - #ATGTGATG                2748                                                                          Asn Asn Asn Leu Glu Leu                                                       225                 2 - #30                                                   #CTG CTG       2802CGAG CTAGCTATAA TTAGGAGCAT ATCAG GTG                       #              Val Leu L - #eu                                                - ACA ACA CCA GCC ATA CCA GAA GAC AGG ATG AA - #G GAG ATC ACC AAG GCT         2850                                                                          Thr Thr Pro Ala Ile Pro Glu Asp Arg Met Ly - #s Glu Ile Thr Lys Ala           #   245                                                                       - TCA GAA GGC TTC GTC TAC CTG GTAGTTATAT GTATATATA - #G ATGGACGACG            2901                                                                          Ser Glu Gly Phe Val Tyr Leu                                                   250                 2 - #55                                                   - TAACTCATTC CAGCCCCATG CATATATGGA GGCTTCAATT CTGCAGAGAC GA - #CGAAGACC       2961                                                                          - ACGACGACGA CTAACACTAG CTAGGGGCGT ACGTTGCAG GTG AGC GTG - # AAC GGA          3015                                                                          #       Val Ser Val Asn Gly                                                   # 260                                                                         - GTG ACA GGT CCT CGC GCA AAC GTG AAC CCA CG - #A GTG GAG TCA CTC ATC         3063                                                                          Val Thr Gly Pro Arg Ala Asn Val Asn Pro Ar - #g Val Glu Ser Leu Ile           #           275                                                               - CAG GAG GTT AAG AAG GTG ACT AAC AAG CCC GT - #T GCT GTT GGC TTC GGC         3111                                                                          Gln Glu Val Lys Lys Val Thr Asn Lys Pro Va - #l Ala Val Gly Phe Gly           #       290                                                                   - ATA TCC AAG CCC GAG CAC GTG AAG CAGGTACGTA CG - #TAGCTGAC CAAAAAAAAC        3165                                                                          Ile Ser Lys Pro Glu His Val Lys                                               #   300                                                                       - TGTTAACAAG TTTTGTTTGA CAAGCCGGCT ACTAGCTAGC TAACAGTGAT CA - #GTGACACA       3225                                                                          - CACACACACA CAG ATT GCG CAG TGG GGC GCT GAC GG - #G GTG ATC ATC GGC          3274                                                                          #Trp Gly Ala Asp Gly Val Ile Ile Gly                                          #     310                                                                     - AGC GCC ATG GTG AGG CAG CTG GGC GAA GCG GC - #T TCT CCC AAG CAA GGC         3322                                                                          Ser Ala Met Val Arg Gln Leu Gly Glu Ala Al - #a Ser Pro Lys Gln Gly           315                 3 - #20                 3 - #25                 3 -       #30                                                                           - CTG AGG AGG CTG GAG GAG TAT GCC AGG GGC AT - #G AAG AAC GCG CTG CCA         3370                                                                          Leu Arg Arg Leu Glu Glu Tyr Ala Arg Gly Me - #t Lys Asn Ala Leu Pro           #               345                                                           - TGAGTCCATG ACAAAGTAAA ACGTACAGAG ACACTTGATA ATATCTATCT AT - #CATCTCGG       3430                                                                          - AGAAGACGAC CGACCAATAA AAATAAGCCA AGTGGAAGTG AAGCTTAGCT GT - #ATATACAC       3490                                                                          - CGTACGTCGT CGTCGTCGTT CCGGATCGAT CTCGGCCGGC TAGCTAGCAG AA - #CGTGTACG       3550                                                                          - TAGTAGTATG TAATGCATGG AGTGTGGAGC TACTAGCTAG CTGGCCGTTC AT - #TCGATTAT       3610                                                                          - AATTCTTCGC TCTGCTGTGG TAGCAGATGT ACCTAGTCGA TCTTGTACGA CG - #AAGAAGCT       3670                                                                          - GGCTAGCTAG CCGTCTCGAT CGTATATGTA CTGATTAATC TGCAGATTGA AT - #AAAAACTA       3730                                                                          - CAGTACGCAT ATGATGCGTA CGTACGTGTG TATAGTTTGT GCTCATATAT GC - #TCCTCATC       3790                                                                          - ACCTGCCTGA TCTGCCCATC GATCTCTCTC GTACTCCTTC CTGTTAAATG CC - #TTCTTTGA       3850                                                                          - CAGACACACC ACCACCAGCA GCAGTGACGC TCTGCACGCC GCCGCTTTAA GA - #CATGTAAG       3910                                                                          - ATATTTTAAG AGGTATAAGA TACCAAGGAG CACAAATCTG GAGCACTGGG AT - #ATTGCAAA       3970                                                                          - GACAAAAAAA AAACAAAATT AAAGTCCCAC CAAAGTAGAG ATAGTAAAGA GG - #TGGATGGA       4030                                                                          - TTAAAATTAT CTCATGATTT TTGGATCTGC TCAAATAGAT CGATATGGTA TT - #CAGATCTA       4090                                                                          - TGTTGTATAG CCTTTTCATT AGCTTTCTGA AAAAAAAATG GTATGATGAG TG - #CGGAGTAG       4150                                                                          - CTAGGGCTGT GAAGGAGTCG GATGGGCTTC CACGTACTTG TTTGTGGCCC TA - #GTCCGGTT       4210                                                                          - CTATTTAGGT CCGATCCGAG TCCGGCATGG TCCGGTTCCA TACGGGCTAG GA - #CCAAGCTC       4270                                                                          - GGCACGTGAG TTTTAGGCCC GTCGGCTAGC CCGAGCACGA CCCGTTTTTA AA - #CTGGCTAG       4330                                                                          - GACTCGCCCA TTTAATAAGA CAAACATTGC AAAAAATAGC TCTATTTTTT AT - #TTAAAATA       4390                                                                          - TATTGTTTAT TTGTGAAATG TGTATTATTT GTAATATATA TTATTGTATA TA - #GTTATATC       4450                                                                          - TTCAATTATG ATTTATAAAT ATGTTTTTTA TTATGAACTC AATTTTAAGT TT - #GATTTATG       4510                                                                          - CGTTGGCGGG CTCGAGGAGG CACGGTGAAC ATTTTTGGGT CGGGCTTAAC GG - #GTCGGCCC       4570                                                                          - GGCCCGGTTC GGCCCATCCA CGGCCCATCC CGTGTCGGCC TCGTTCGGTG AG - #TTCAGCCC       4630                                                                          - GTCGGACAAC CCGTCCCCGG CCCGGATAAT TAATCGGGCC TAACCGTGGC GT - #GCTTAAAC       4690                                                                          - GGTCCGTGCC TCAACGGACC GGGCCGCGGG CGGCCCGTTT GACATCTCTA GT - #GGTGTGAT       4750                                                                          - TAGAGATGGC GATGGGAACC GATCACTGAT TCCGTGTGGA GAATTCGATA TC - #AAGCTTAT       4810                                                                          #        4817                                                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 346 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Met Ala Phe Ala Pro Lys Thr Ser Ser Ser Se - #r Ser Leu Ser Ser Ala         #                 15                                                          - Leu Gln Ala Ala Gln Ser Pro Pro Leu Leu Le - #u Arg Arg Met Ser Ser         #             30                                                              - Thr Ala Thr Pro Arg Arg Arg Tyr Asp Ala Al - #a Val Val Val Thr Thr         #         45                                                                  - Thr Thr Thr Ala Arg Ala Ala Ala Ala Ala Va - #l Thr Val Pro Ala Ala         #     60                                                                      - Pro Pro Gln Ala Gly Arg Arg Arg Arg Cys Hi - #s Gln Ser Lys Arg Arg         # 80                                                                          - His Pro Gln Arg Arg Ser Arg Pro Val Ser As - #p Thr Met Ala Ala Leu         #                 95                                                          - Met Ala Lys Gly Lys Thr Ala Phe Ile Pro Ty - #r Ile Thr Ala Gly Asp         #           110                                                               - Pro Asp Leu Ala Thr Thr Ala Glu Ala Leu Ar - #g Leu Leu Asp Gly Cys         #       125                                                                   - Gly Ala Asp Val Ile Glu Leu Gly Val Pro Cy - #s Ser Asp Pro Tyr Ile         #   140                                                                       - Asp Gly Pro Ile Ile Gln Ala Ser Val Ala Ar - #g Ala Leu Ala Ser Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Thr Met Asp Ala Val Leu Glu Met Leu Ar - #g Glu Val Thr Pro Glu         #               175                                                           - Leu Ser Cys Pro Val Val Leu Leu Ser Tyr Ty - #r Lys Pro Ile Met Ser         #           190                                                               - Arg Ser Leu Ala Glu Met Lys Glu Ala Gly Va - #l His Gly Leu Ile Val         #       205                                                                   - Pro Asp Leu Pro Tyr Val Ala Ala His Ser Le - #u Trp Ser Glu Ala Lys         #   220                                                                       - Asn Asn Asn Leu Glu Leu Val Leu Leu Thr Th - #r Pro Ala Ile Pro Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Arg Met Lys Glu Ile Thr Lys Ala Ser Gl - #u Gly Phe Val Tyr Leu         #               255                                                           - Val Ser Val Asn Gly Val Thr Gly Pro Arg Al - #a Asn Val Asn Pro Arg         #           270                                                               - Val Glu Ser Leu Ile Gln Glu Val Lys Lys Va - #l Thr Asn Lys Pro Val         #       285                                                                   - Ala Val Gly Phe Gly Ile Ser Lys Pro Glu Hi - #s Val Lys Gln Ile Ala         #   300                                                                       - Gln Trp Gly Ala Asp Gly Val Ile Ile Gly Se - #r Ala Met Val Arg Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Gly Glu Ala Ala Ser Pro Lys Gln Gly Le - #u Arg Arg Leu Glu Glu         #               335                                                           - Tyr Ala Arg Gly Met Lys Asn Ala Leu Pro                                     #           345                                                               - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1349 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..1226                                               #/note= "cDNA sequence for maize                                                             pollen-speci - #fic calcium dependent protein kinase gene      as                                                                            #in Figure 30."disclosed                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - TG CAG ATC ATG CAC CAC CTC TCC GGC CAG CCC - # AAC GTG GTG GGC CTC            47                                                                          #Pro Asn Val Val Gly Leueu Ser Gly Gln                                        #              360                                                            - CGC GGC GCG TAC GAG GAC AAG CAG AGC GTG CA - #C CTC GTC ATG GAG CTG           95                                                                          Arg Gly Ala Tyr Glu Asp Lys Gln Ser Val Hi - #s Leu Val Met Glu Leu           #           375                                                               - TGC GCG GGC GGG GAG CTC TTC GAC CGC ATC AT - #C GCC CGG GGC CAG TAC          143                                                                          Cys Ala Gly Gly Glu Leu Phe Asp Arg Ile Il - #e Ala Arg Gly Gln Tyr           #       390                                                                   - ACG GAG CGC GGC GCC GCG GAG CTG CTG CGC GC - #C ATC GTG CAG ATC GTG          191                                                                          Thr Glu Arg Gly Ala Ala Glu Leu Leu Arg Al - #a Ile Val Gln Ile Val           #   405                                                                       - CAC ACC TGC CAC TCC ATG GGG GTG ATG CAC CG - #G GAC ATC AAG CCC GAG          239                                                                          His Thr Cys His Ser Met Gly Val Met His Ar - #g Asp Ile Lys Pro Glu           410                 4 - #15                 4 - #20                 4 -       #25                                                                           - AAC TTC CTG CTG CTC AGC AAG GAC GAG GAC GC - #G CCG CTC AAG GCC ACC          287                                                                          Asn Phe Leu Leu Leu Ser Lys Asp Glu Asp Al - #a Pro Leu Lys Ala Thr           #               440                                                           - GAC TTC GGC CTC TCC GTC TTC TTC AAG GAG GG - #C GAG CTG CTC AGG GAC          335                                                                          Asp Phe Gly Leu Ser Val Phe Phe Lys Glu Gl - #y Glu Leu Leu Arg Asp           #           455                                                               - ATC GTC GGC AGC GCC TAC TAC ATC GCG CCC GA - #G GTG CTC AAG AGG AAG          383                                                                          Ile Val Gly Ser Ala Tyr Tyr Ile Ala Pro Gl - #u Val Leu Lys Arg Lys           #       470                                                                   - TAC GGC CCG GAG GCC GAC ATC TGG AGC GTC GG - #C GTC ATG CTC TAC ATC          431                                                                          Tyr Gly Pro Glu Ala Asp Ile Trp Ser Val Gl - #y Val Met Leu Tyr Ile           #   485                                                                       - TTC CTC GCC GGC GTG CCT CCC TTC TGG GCA GA - #G AAC GAG AAC GGC ATC          479                                                                          Phe Leu Ala Gly Val Pro Pro Phe Trp Ala Gl - #u Asn Glu Asn Gly Ile           490                 4 - #95                 5 - #00                 5 -       #05                                                                           - TTC ACC GCC ATC CTG CGA GGG CAG CTT GAC CT - #C TCC AGC GAG CCA TGG          527                                                                          Phe Thr Ala Ile Leu Arg Gly Gln Leu Asp Le - #u Ser Ser Glu Pro Trp           #               520                                                           - CCA CAC ATC TCG CCG GGA GCC AAG GAT CTC GT - #C AAG AAG ATG CTC AAC          575                                                                          Pro His Ile Ser Pro Gly Ala Lys Asp Leu Va - #l Lys Lys Met Leu Asn           #           535                                                               - ATC AAC CCC AAG GAG CGG CTC ACG GCG TTC CA - #G GTC CTC AAT CAC CCA          623                                                                          Ile Asn Pro Lys Glu Arg Leu Thr Ala Phe Gl - #n Val Leu Asn His Pro           #       550                                                                   - TGG ATC AAA GAA GAC GGA GAC GCG CCT GAC AC - #G CCG CTT GAC AAC GTT          671                                                                          Trp Ile Lys Glu Asp Gly Asp Ala Pro Asp Th - #r Pro Leu Asp Asn Val           #   565                                                                       - GTT CTC GAC AGG CTC AAG CAG TTC AGG GCC AT - #G AAC CAG TTC AAG AAA          719                                                                          Val Leu Asp Arg Leu Lys Gln Phe Arg Ala Me - #t Asn Gln Phe Lys Lys           570                 5 - #75                 5 - #80                 5 -       #85                                                                           - GCA GCA TTG AGG ATC ATA GCT GGG TGC CTA TC - #C GAA GAG GAG ATC ACA          767                                                                          Ala Ala Leu Arg Ile Ile Ala Gly Cys Leu Se - #r Glu Glu Glu Ile Thr           #               600                                                           - GGG CTG AAG GAG ATG TTC AAG AAC ATT GAC AA - #G GAT AAC AGC GGG ACC          815                                                                          Gly Leu Lys Glu Met Phe Lys Asn Ile Asp Ly - #s Asp Asn Ser Gly Thr           #           615                                                               - ATT ACC CTC GAC GAG CTC AAA CAC GGG TTG GC - #A AAG CAC GGG CCC AAG          863                                                                          Ile Thr Leu Asp Glu Leu Lys His Gly Leu Al - #a Lys His Gly Pro Lys           #       630                                                                   - CTG TCA GAC AGC GAA ATG GAG AAA CTA ATG GA - #A GCA GCT GAC GCT GAC          911                                                                          Leu Ser Asp Ser Glu Met Glu Lys Leu Met Gl - #u Ala Ala Asp Ala Asp           #   645                                                                       - GGC AAC GGG TTA ATT GAC TAC GAC GAA TTC GT - #C ACC GCA ACA GTG CAT          959                                                                          Gly Asn Gly Leu Ile Asp Tyr Asp Glu Phe Va - #l Thr Ala Thr Val His           650                 6 - #55                 6 - #60                 6 -       #65                                                                           - ATG AAC AAA CTG GAT AGA GAA GAG CAC CTT TA - #C ACA GCA TTC CAG TAT         1007                                                                          Met Asn Lys Leu Asp Arg Glu Glu His Leu Ty - #r Thr Ala Phe Gln Tyr           #               680                                                           - TTC GAC AAG GAC AAC AGC GGG TAC ATT ACT AA - #A GAA GAG CTT GAG CAC         1055                                                                          Phe Asp Lys Asp Asn Ser Gly Tyr Ile Thr Ly - #s Glu Glu Leu Glu His           #           695                                                               - GCC TTG AAG GAG CAA GGG TTG TAT GAC GCC GA - #T AAA ATC AAA GAC ATC         1103                                                                          Ala Leu Lys Glu Gln Gly Leu Tyr Asp Ala As - #p Lys Ile Lys Asp Ile           #       710                                                                   - ATC TCC GAT GCC GAC TCT GAC AAT GAT GGA AG - #G ATA GAT TAT TCA GAG         1151                                                                          Ile Ser Asp Ala Asp Ser Asp Asn Asp Gly Ar - #g Ile Asp Tyr Ser Glu           #   725                                                                       - TTT GTG GCG ATG ATG AGG AAA GGG ACG GCT GG - #T GCC GAG CCA ATG AAC         1199                                                                          Phe Val Ala Met Met Arg Lys Gly Thr Ala Gl - #y Ala Glu Pro Met Asn           730                 7 - #35                 7 - #40                 7 -       #45                                                                           - ATC AAG AAG AGG CGA GAC ATA GTC CTA TAGTGAAGT - #G AAGCAGCAAG               1246                                                                          Ile Lys Lys Arg Arg Asp Ile Val Leu                                                           750                                                           - TGTGTAATGT AATGTGTATA GCAGCTCAAA CAAGCAAATT TGTACATCTG TA - #CACAAATG       1306                                                                          #                 134 - #9AAAAAAAA AAAAAAAAAA AAA                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 408 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Gln Ile Met His His Leu Ser Gly Gln Pro As - #n Val Val Gly Leu Arg         #                 15                                                          - Gly Ala Tyr Glu Asp Lys Gln Ser Val His Le - #u Val Met Glu Leu Cys         #             30                                                              - Ala Gly Gly Glu Leu Phe Asp Arg Ile Ile Al - #a Arg Gly Gln Tyr Thr         #         45                                                                  - Glu Arg Gly Ala Ala Glu Leu Leu Arg Ala Il - #e Val Gln Ile Val His         #     60                                                                      - Thr Cys His Ser Met Gly Val Met His Arg As - #p Ile Lys Pro Glu Asn         # 80                                                                          - Phe Leu Leu Leu Ser Lys Asp Glu Asp Ala Pr - #o Leu Lys Ala Thr Asp         #                 95                                                          - Phe Gly Leu Ser Val Phe Phe Lys Glu Gly Gl - #u Leu Leu Arg Asp Ile         #           110                                                               - Val Gly Ser Ala Tyr Tyr Ile Ala Pro Glu Va - #l Leu Lys Arg Lys Tyr         #       125                                                                   - Gly Pro Glu Ala Asp Ile Trp Ser Val Gly Va - #l Met Leu Tyr Ile Phe         #   140                                                                       - Leu Ala Gly Val Pro Pro Phe Trp Ala Glu As - #n Glu Asn Gly Ile Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Ala Ile Leu Arg Gly Gln Leu Asp Leu Se - #r Ser Glu Pro Trp Pro         #               175                                                           - His Ile Ser Pro Gly Ala Lys Asp Leu Val Ly - #s Lys Met Leu Asn Ile         #           190                                                               - Asn Pro Lys Glu Arg Leu Thr Ala Phe Gln Va - #l Leu Asn His Pro Trp         #       205                                                                   - Ile Lys Glu Asp Gly Asp Ala Pro Asp Thr Pr - #o Leu Asp Asn Val Val         #   220                                                                       - Leu Asp Arg Leu Lys Gln Phe Arg Ala Met As - #n Gln Phe Lys Lys Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Leu Arg Ile Ile Ala Gly Cys Leu Ser Gl - #u Glu Glu Ile Thr Gly         #               255                                                           - Leu Lys Glu Met Phe Lys Asn Ile Asp Lys As - #p Asn Ser Gly Thr Ile         #           270                                                               - Thr Leu Asp Glu Leu Lys His Gly Leu Ala Ly - #s His Gly Pro Lys Leu         #       285                                                                   - Ser Asp Ser Glu Met Glu Lys Leu Met Glu Al - #a Ala Asp Ala Asp Gly         #   300                                                                       - Asn Gly Leu Ile Asp Tyr Asp Glu Phe Val Th - #r Ala Thr Val His Met         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Lys Leu Asp Arg Glu Glu His Leu Tyr Th - #r Ala Phe Gln Tyr Phe         #               335                                                           - Asp Lys Asp Asn Ser Gly Tyr Ile Thr Lys Gl - #u Glu Leu Glu His Ala         #           350                                                               - Leu Lys Glu Gln Gly Leu Tyr Asp Ala Asp Ly - #s Ile Lys Asp Ile Ile         #       365                                                                   - Ser Asp Ala Asp Ser Asp Asn Asp Gly Arg Il - #e Asp Tyr Ser Glu Phe         #   380                                                                       - Val Ala Met Met Arg Lys Gly Thr Ala Gly Al - #a Glu Pro Met Asn Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Lys Lys Arg Arg Asp Ile Val Leu                                                             405                                                           - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 464 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..464                                                #/note= "derived protein sequence of                                                         pollen sp - #ecific CDPK as disclosed in Figure 34."           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Val Leu Gly Arg Pro Met Glu Asp Val Arg Al - #a Thr Tyr Ser Met Gly         #                15                                                           - Lys Glu Leu Gly Arg Gly Gln Phe Gly Val Th - #r His Leu Cys Thr His         #            30                                                               - Arg Thr Ser Gly Glu Lys Leu Ala Cys Lys Th - #r Ile Ala Lys Arg Lys         #        45                                                                   - Leu Ala Ala Arg Glu Asp Val Asp Asp Val Ar - #g Arg Glu Val Gln Ile         #    60                                                                       - Met His His Leu Ser Gly Gln Pro Asn Val Va - #l Gly Leu Arg Gly Ala         #80                                                                           - Tyr Glu Asp Lys Gln Ser Val His Leu Val Me - #t Glu Leu Cys Ala Gly         #                95                                                           - Gly Glu Leu Phe Asp Arg Ile Ile Ala Arg Gl - #y Gln Tyr Thr Glu Arg         #           110                                                               - Gly Ala Ala Glu Leu Leu Arg Ala Ile Val Gl - #n Ile Val His Thr Cys         #       125                                                                   - His Ser Met Gly Val Met His Arg Asp Ile Ly - #s Pro Glu Asn Phe Leu         #   140                                                                       - Leu Leu Ser Lys Asp Glu Asp Ala Pro Leu Ly - #s Ala Thr Asp Phe Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Ser Val Phe Phe Lys Glu Gly Glu Leu Le - #u Arg Asp Ile Val Gly         #               175                                                           - Ser Ala Tyr Tyr Ile Ala Pro Glu Val Leu Ly - #s Arg Lys Tyr Gly Pro         #           190                                                               - Glu Ala Asp Ile Trp Ser Val Gly Val Met Le - #u Tyr Ile Phe Leu Ala         #       205                                                                   - Gly Val Pro Pro Phe Trp Ala Glu Asn Glu As - #n Gly Ile Phe Thr Ala         #   220                                                                       - Ile Leu Arg Gly Gln Leu Asp Leu Ser Ser Gl - #u Pro Trp Pro His Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Pro Gly Ala Lys Asp Leu Val Lys Lys Me - #t Leu Asn Ile Asn Pro         #               255                                                           - Lys Glu Arg Leu Thr Ala Phe Gln Val Leu As - #n His Pro Trp Ile Lys         #           270                                                               - Glu Asp Gly Asp Ala Pro Asp Thr Pro Leu As - #p Asn Val Val Leu Asp         #       285                                                                   - Arg Leu Lys Gln Phe Arg Ala Met Asn Gln Ph - #e Lys Lys Ala Ala Leu         #   300                                                                       - Arg Ile Ile Ala Gly Cys Leu Ser Glu Glu Gl - #u Ile Thr Gly Leu Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Met Phe Lys Asn Ile Asp Lys Asp Asn Se - #r Gly Thr Ile Thr Leu         #               335                                                           - Asp Glu Leu Lys His Gly Leu Ala Lys His Gl - #y Pro Lys Leu Ser Asp         #           350                                                               - Ser Glu Met Glu Lys Leu Met Glu Ala Ala As - #p Ala Asp Gly Asn Gly         #       365                                                                   - Leu Ile Asp Tyr Asp Glu Phe Val Thr Ala Th - #r Val His Met Asn Lys         #   380                                                                       - Leu Asp Arg Glu Glu His Leu Tyr Thr Ala Ph - #e Gln Tyr Phe Asp Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Asn Ser Gly Tyr Ile Thr Lys Glu Glu Le - #u Glu His Ala Leu Lys         #               415                                                           - Glu Gln Gly Leu Tyr Asp Ala Asp Lys Ile Ly - #s Asp Ile Ile Ser Asp         #           430                                                               - Ala Asp Ser Asp Asn Asp Gly Arg Ile Asp Ty - #r Ser Glu Phe Val Ala         #       445                                                                   - Met Met Arg Lys Gly Thr Ala Gly Ala Glu Pr - #o Met Asn Ile Lys Lys         #   460                                                                       - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 295 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..295                                                #/note= "rat protein kinase IIN:                                                             protein s - #equence as shown in Figure 32."                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Tyr Gln Leu Phe Glu Glu Leu Gly Lys Gly Al - #a Phe Ser Val Val Arg         #                15                                                           - Arg Cys Val Lys Lys Thr Ser Thr Gln Glu Ty - #r Ala Ala Lys Ile Ile         #            30                                                               - Asn Thr Lys Lys Leu Ser Ala Arg Asp His Gl - #n Lys Leu Glu Arg Glu         #        45                                                                   - Ala Arg Ile Cys Arg Leu Leu Lys His Pro As - #n Ile Val Arg Leu His         #    60                                                                       - Asp Ser Ile Ser Glu Glu Gly Phe His Tyr Le - #u Val Phe Asp Leu Val         #80                                                                           - Thr Gly Gly Glu Leu Phe Glu Asp Ile Val Al - #a Arg Glu Tyr Tyr Ser         #                95                                                           - Glu Ala Asp Ala Ser His Cys Ile His Gln Il - #e Leu Glu Ser Val Asn         #           110                                                               - His Ile His Gln His Asp Ile Val His Arg As - #p Leu Lys Pro Glu Asn         #       125                                                                   - Leu Leu Leu Ala Ser Lys Cys Lys Gly Ala Al - #a Val Lys Leu Ala Asp         #   140                                                                       - Phe Gly Leu Ala Ile Glu Val Gln Gly Glu Gl - #n Gln Ala Trp Phe Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Ala Gly Thr Pro Gly Tyr Leu Ser Pro Gl - #u Val Leu Arg Lys Asp         #               175                                                           - Pro Tyr Gly Lys Pro Val Asp Ile Trp Ala Cy - #s Gly Val Ile Leu Tyr         #           190                                                               - Ile Leu Leu Val Gly Tyr Pro Pro Phe Trp As - #p Glu Asp Gln His Lys         #       205                                                                   - Leu Tyr Gln Gln Ile Lys Ala Gly Ala Tyr As - #p Phe Pro Ser Pro Glu         #   220                                                                       - Trp Asp Thr Val Thr Pro Glu Ala Lys Asn Le - #u Ile Asn Gln Met Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Ile Asn Pro Ala Lys Arg Ile Thr Ala As - #p Gln Ala Leu Lys His         #               255                                                           - Pro Trp Val Cys Gln Arg Ser Thr Val Ala Se - #r Met Met His Arg Gln         #           270                                                               - Glu Thr Val Glu Cys Leu Arg Lys Phe Asn Al - #a Arg Arg Lys Leu Lys         #       285                                                                   - Gly Ala Ile Leu Thr Thr Met                                                 #   295                                                                       - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 142 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..142                                                #/note= "human calmodulin protein                                             #as shown in Figure 33."                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Leu Thr Glu Glu Gln Ile Ala Glu Phe Lys Gl - #u Ala Phe Ser Leu Phe         #                15                                                           - Asp Lys Asp Gly Asp Gly Thr Ile Thr Thr Ly - #s Glu Leu Gly Thr Val         #            30                                                               - Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Al - #a Glu Leu Gln Asp Met         #        45                                                                   - Ile Asn Glu Val Asp Ala Asp Gly Asn Gly Th - #r Ile Asp Phe Pro Glu         #    60                                                                       - Phe Leu Thr Met Met Ala Arg Lys Met Lys As - #p Thr Asp Ser Glu Glu         #80                                                                           - Glu Ile Arg Glu Ala Phe Arg Val Lys Asp Ly - #s Asp Gly Asn Gly Tyr         #                95                                                           - Ile Ser Ala Ala Glu Leu Arg His Val Met Th - #r Asn Leu Gly Glu Lys         #           110                                                               - Leu Thr Asp Glu Glu Val Asp Glu Met Ile Ar - #g Glu Ala Asp Ile Asp         #       125                                                                   - Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Va - #l Gln Met Met                 #   140                                                                       - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 463 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..463                                                #/note= "protein sequence forON:                                                             soybean C - #DPK as shown in Figure 34."                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Val Leu Pro Gln Arg Thr Gln Asn Ile Arg Gl - #u Val Tyr Glu Val Gly         #                15                                                           - Arg Lys Leu Gly Gln Gly Gln Phe Gly Thr Th - #r Phe Glu Cys Thr Arg         #            30                                                               - Arg Ala Ser Gly Gly Lys Phe Ala Cys Lys Se - #r Ile Pro Lys Arg Lys         #        45                                                                   - Leu Leu Cys Lys Glu Asp Tyr Glu Asp Val Tr - #p Arg Glu Ile Gln Ile         #    60                                                                       - Met His His Leu Ser Glu His Ala Asn Val Va - #l Arg Ile Glu Gly Thr         #80                                                                           - Tyr Glu Asp Ser Thr Ala Val His Leu Val Me - #t Glu Leu Cys Glu Gly         #                95                                                           - Gly Glu Leu Phe Asp Arg Ile Val Gln Lys Gl - #y His Tyr Ser Glu Arg         #           110                                                               - Gln Ala Ala Arg Leu Ile Lys Thr Ile Val Gl - #u Val Val Glu Ala Cys         #       125                                                                   - His Ser Leu Gly Val Met His Arg Asp Leu Ly - #s Pro Glu Asn Phe Leu         #   140                                                                       - Phe Asp Thr Ile Asp Glu Asp Ala Lys Leu Ly - #s Ala Thr Asp Phe Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Ser Val Phe Tyr Lys Pro Gly Glu Ser Ph - #e Cys Asp Val Val Gly         #               175                                                           - Ser Pro Tyr Tyr Val Ala Pro Glu Val Leu Ar - #g Lys Leu Tyr Gly Pro         #           190                                                               - Glu Ser Asp Val Trp Ser Ala Gly Val Ile Le - #u Tyr Ile Leu Leu Ser         #       205                                                                   - Gly Val Pro Pro Phe Trp Ala Glu Ser Glu Pr - #o Gly Ile Phe Arg Gln         #   220                                                                       - Ile Leu Leu Gly Lys Leu Asp Phe His Ser Gl - #u Pro Trp Pro Ser Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Asp Ser Ala Lys Asp Leu Ile Arg Lys Me - #t Leu Asp Gln Asn Pro         #               255                                                           - Lys Thr Arg Leu Thr Ala His Glu Val Leu Ar - #g His Pro Trp Ile Val         #           270                                                               - Asp Asp Asn Ile Ala Pro Asp Lys Pro Leu As - #p Ser Ala Val Leu Ser         #       285                                                                   - Arg Leu Lys Gln Phe Ser Ala Met Asn Lys Le - #u Lys Lys Met Ala Leu         #   300                                                                       - Arg Val Ile Ala Glu Arg Leu Ser Glu Glu Gl - #u Ile Gly Gly Leu Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Leu Phe Lys Met Ile Asp Thr Asp Asn Se - #r Gly Thr Ile Thr Phe         #               335                                                           - Asp Glu Leu Lys Asp Gly Leu Lys Arg Val Gl - #y Ser Glu Leu Met Glu         #           350                                                               - Ser Glu Ile Lys Asp Leu Met Asp Ala Ala As - #p Ile Asp Lys Ser Gly         #       365                                                                   - Thr Ile Asp Tyr Gly Glu Phe Ile Ala Ala Th - #r Val His Leu Asn Lys         #   380                                                                       - Leu Glu Arg Glu Glu Asn Leu Val Ser Ala Ph - #e Ser Tyr Phe Asp Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Gly Ser Gly Tyr Ile Thr Leu Asp Glu Il - #e Gln Gln Ala Cys Lys         #               415                                                           - Asp Phe Gly Leu Asp Asp Ile His Ile Asp As - #p Met Ile Lys Glu Ile         #           430                                                               - Asp Gln Asp Asn Asp Gly Gln Ile Asp Tyr Gl - #y Glu Phe Ala Ala Met         #       445                                                                   - Met Arg Lys Gly Asn Gly Gly Ile Gly Arg Ar - #g Thr Met Arg Lys             #   460                                                                       - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 4162 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1418..1427                                            #/note= "start of mRNA"ORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 1481..2366                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 2367..2451                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 2452..2602                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 2603..2690                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 2691..2804                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 2805..2906                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 2907..3075                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 3076..3177                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 3178..3304                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 3305..3398                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 3399..3498                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 3499..3713                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: exon                                                            (B) LOCATION: 3714..3811                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: promoter                                                        (B) LOCATION: 1..1477                                                         (C) IDENTIFICATION METHOD: - # experimental                         #/partial (D) OTHER INFORMATION:                                                             /function=- # "pollen-specific promoter region"                               /evidence=- # EXPERIMENTAL                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - TTAGTAACAC CTCTCCAATC GCTTGGGTTG GCACATTCTT AGCTTTTATC AC - #ATTTTAAG         60                                                                          - AAATAGAGTT CACCACCTTC AAAATAATGC CTATACAATG AATGATCGCT TG - #GATGCAAT        120                                                                          - ATAGCTAGAT TCAACTAGCT ATATATGGTC AATAGAACCC TGTGAGCACC TC - #ACAAACAC        180                                                                          - GACTTCAATT TTGAGACCCT AAGCGAGTAA ATGGTTAAAG TCCTCTTATT AT - #TAGTCTTA        240                                                                          - GGACTTCTCC TTGCTAAATG CTTGTCAGCG ATCTATATAT CTTCCCCACT GC - #GGGAGATA        300                                                                          - CTATATATAG GGCCTTGGAC CTCTAGGGTA TCTCAAAGGC CTAGTCACAA CA - #ATTCTCAA        360                                                                          - CAGTATTTAA TTTTATACAT GTATGAACAG TGTAGGAATT TGAGTGCCCA AC - #CCAAGAGT        420                                                                          - GGGAGGTGTA AATTGGGTAG CTAAACTTAA ATAGGGCTCT TCTTATTTAG GT - #TTATCTAG        480                                                                          - TCTCTACTTA GACTAATTCA GAAAGAATTT TACAACCTAT GGTTAATCAT AT - #CTCTAGTC        540                                                                          - TAAGCAAATT TAGGAAAGTT AAAAGCACAC AATTAGGCAC ATGTGAAAGA TG - #TGTATGGT        600                                                                          - AAGTAAAAGA CTTATAAGGA AAAAGTGGGT GAATCCTCAA GATGTGGTGG TA - #TATCCCAA        660                                                                          - TGATATTAGA TGCCAGAATA TAGGGGGGAA ATCGATGTAT ACCATCTCTA CC - #AGGATACC        720                                                                          - TGTGCGGACT GTGCAACTGA CACATGGACC ATGGTGTCTT CTTAGATTTG GT - #TATTAGCT        780                                                                          - AATTGCGCTA CAACTTGTTC AAGGCTAGAC CAAATTAAAA AACTAATATT AA - #ACATAAAA        840                                                                          - AGTTAGGCAA ACTATAGTAA ATTATGCAGC GATCCAACAA CAAGCCATGT CT - #CGTGGGTC        900                                                                          - ATGAGCCACG CGTCGGCCAT ACACCCACAT GATGTTTCCA TACGGATGGT CC - #TTATGCAA        960                                                                          - TTTTGTCTGC AAAACACAAG CCTTAATACA GCCACGCGAC AATCATGGAA GT - #GGTCGTTT       1020                                                                          - TAGGTCCTCA TCATGAAGTT CAGGGAAAAC GCATCAAATG TAATGCAGAG AA - #ATGGTATT       1080                                                                          - TCTTCTCTTG TAAATCAGGG AGAGGAGTAC CATCAGTACA GATTCAGAAT CA - #GAATTCAG       1140                                                                          - TCTTCCAACG ACAATAATCG CAGCATCTTG TAAAAATTTG CAGAAACTTC TG - #TTTGACTT       1200                                                                          - GTAGCCCTGA CCTTTGCAAA TATTTGAAGT TGTGCCTGCT GACACAACTT CA - #ATCTGGAA       1260                                                                          - GTGCTGTTGA TCAGTTTTGC CAGAAACAGC AAGCAGCCTA TATATATCTG TC - #ACGAGACA       1320                                                                          - CCCTGCCGCC CTCTTCTTTC CCGCCATTCC CTCCCTACCC TTCAAAATCT AG - #AAACCTTT       1380                                                                          - TTTTTTCCTC CCGATACGCC CCTCCATCTC TCGCCGTTCA TGTCCGTGGC TG - #GCTGCCCT       1440                                                                          - CCGTGGGAGC AGGCGGCCGC ACTCGTTCCC CGCCGCAGCC ATGGGCCAGT GC - #TGCTCCAA       1500                                                                          - GGGCGCCGGA GAGGCCCCGC CACCGAGGCG CCAAACGGCA GGCGCCAAGC CG - #CGGGCGTC       1560                                                                          - CGCGAACAAC GCCGACGGAC AACGGGCGTC GTCCTCGTCC GCGGTGGCTG CT - #GCCGCTGC       1620                                                                          - TGCTGCCGGT GGTGGTGGCG GCGGCACGAC GAAGCCGGCC TCACCCACCG GC - #GGCGCCAG       1680                                                                          - GGCCAGCTCC GGCAGCAAAC CGGCGGCGGC CGTGGGCACG GTGCTGGGCC GG - #CCCATGGA       1740                                                                          - GGACGTGCGC GCGACCTACT CGATGGGCAA GGAGCTCGGG CGCGGGCAGT TC - #GGCGTGAC       1800                                                                          - GCACCTGTGC ACGCACCGGA CGAGCGGCGA GAAGCTGGCG TGCAAGACGA TC - #GCGAAGCG       1860                                                                          - GAAGCTGGCG GCCAGGGAGG ACGTGGACGA CGTGCGGCGG GAGGTGCAGA TC - #ATGCACCA       1920                                                                          - CCTCTCCGGC CAGCCCAACG TGGTGGGCCT CCGCGGCGCG TACGAGGACA AG - #CAGAGCGT       1980                                                                          - GCACCTCGTC ATGGAGCTGT GCGCGGGCGG GGAGCTCTTC GACCGCATCA TC - #GCCCGGGG       2040                                                                          - CCAGTACACG GAGCGCGGCG CCGCGGAGCT GCTGCGCGCC ATCGTGCAGA TC - #GTGCACAC       2100                                                                          - CTGCCACTCC ATGGGGGTGA TGCACCGGGA CATCAAGCCC GAGAACTTCC TG - #CTGCTCAG       2160                                                                          - CAAGGACGAG GACGCGCCGC TCAAGGCCAC CGACTTCGGC CTCTCCGTCT TC - #TTCAAGGA       2220                                                                          - GGGCGAGCTG CTCAGGGACA TCGTCGGCAG CGCCTACTAC ATCGCGCCCG AG - #GTGCTCAA       2280                                                                          - GAGGAAGTAC GGCCCGGAGG CCGACATCTG GAGCGTCGGC GTCATGCTCT AC - #ATCTTCCT       2340                                                                          - CGCCGGCGTG CCTCCCTTCT GGGCAGGTCG GATCCGTCCG TGTTCGTCCT AG - #ACGATATA       2400                                                                          - CAGAACCCGA CGATGGATTT GCTTCTCAGC CCTGTTCTTG CATCACCAGA GA - #ACGAGAAC       2460                                                                          - GGCATCTTCA CCGCCATCCT GCGAGGGCAG CTTGACCTCT CCAGCGAGCC AT - #GGCCACAC       2520                                                                          - ATCTCGCCGG GAGCCAAGGA TCTCGTCAAG AAGATGCTCA ACATCAACCC CA - #AGGAGCGG       2580                                                                          - CTCACGGCGT TCCAGGTCCT CAGTAAGTAC CCAGATCGTT GCTGTCATAC AC - #TCATATGA       2640                                                                          - ATTGTATCGT TCATGAGCAA CGATCGAGCG GATTTGGTGA ACTTGTAGAT CA - #CCCATGGA       2700                                                                          - TCAAAGAAGA CGGAGACGCG CCTGACACGC CGCTTGACAA CGTTGTTCTC GA - #CAGGCTCA       2760                                                                          - AGCAGTTCAG GGCCATGAAC CAGTTCAAGA AAGCAGCATT GAGGGTACAT TA - #TCTGATAA       2820                                                                          - AAGCTCCACA AATACAACTT CTGAAGAACA GCAATGCTTA CACGGCAGAA TT - #TTCATTAT       2880                                                                          - AAATGCTCTT GATGACATAA TGTTAGATCA TAGCTGGGTG CCTATCCGAA GA - #GGAGATCA       2940                                                                          - CAGGGCTGAA GGAGATGTTC AAGAACATTG ACAAGGATAA CAGCGGGACC AT - #TACCCTCG       3000                                                                          - ACGAGCTCAA ACACGGGTTG GCAAAGCACG GGCCCAAGCT GTCAGACAGC GA - #AATGGAGA       3060                                                                          - AACTAATGGA AGCAGTGAGT TTTCAGAGTA CAATCTTAAA AAAAGGAATT GT - #GATTCTTT       3120                                                                          - TCAAAATGAA GAAGTAATCT GAAAACATCC CTGCTGAAAT GCTTTATACA TT - #TCCAGGCT       3180                                                                          - GACGCTGACG GCAACGGGTT AATTGACTAC GACGAATTCG TCACCGCAAC AG - #TGCATATG       3240                                                                          - AACAAACTGG ATAGAGAAGA GCACCTTTAC ACAGCATTCC AGTATTTCGA CA - #AGGACAAC       3300                                                                          - AGCGGGTAAG TTGAACGTTA AAATGATACA GCTGGTACCT GAATTCTGGA CA - #ACACATAT       3360                                                                          - CATAACAGGA CACATATATA ATTCGTTTAT CTCACAGGTA CATTACTAAA GA - #AGAGCTTG       3420                                                                          - AGCACGCCTT GAAGGAGCAA GGGTTGTATG ACGCCGATAA AATCAAAGAC AT - #CATCTCCG       3480                                                                          - ATGCCGACTC TGACAATGTA AGGAACAAAC ATTATTTAAA TTTCAGCCGA CA - #AACTAAAC       3540                                                                          - TATAGAAACC ACATCATGAT ATCAAATTTT GAGGTGGCGG TGCTACAGAA AT - #AGAACCCA       3600                                                                          - GTACACCAAA ATGACTAACT TGTCATGATT AGTTGTTCCT CGTAACTGAA CA - #TTTGTGTT       3660                                                                          - CTTAGTTTCT TATTGTTAAA CCAAAGACTT AAATTCACTT TTGCACATGC AG - #GATGGAAG       3720                                                                          - GATAGATTAT TCAGAGTTTG TGGCGATGAT GAGGAAAGGG ACGGCTGGTG CC - #GAGCCAAT       3780                                                                          - GAACATCAAG AAGAGGCGAG ACATAGTCCT ATAGTGAAGT GAAGCAGAAG TG - #TGTAATGT       3840                                                                          - AATGTGTATA GCAGCTCAAA CAAGCAAATT TGTACATCTG TACACAAATG CA - #ATGGGGTT       3900                                                                          - ACTTTTGCAA CTTAGTTCAT GGATGGTTGT GTACGTTGTG CTATTGATTG CA - #AGTGATTT       3960                                                                          - GAAAGACATG CATACTTAGG AACTGAGAAA GATAGATCTA CTACTGCTAG AG - #ACAGAACA       4020                                                                          - ATAGGATAAT TCAGAAGTGG TATTTCAGAA GACTACAGCT GGCATCTATT AT - #TCTCATTG       4080                                                                          - TCCTCGCAAA AATACTGATG ATGCATTTGA GAGAACAATA TGCAACAAGA TC - #GAGCTCCC       4140                                                                          #               4162GGC CA                                                    - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3546 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Synthetic DNA"RIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3543                                               #/product= "Full-length, hybrid:                                                             maize opt - #imized heat stable cryIA(b)"                      #"DNA sequence as disclosed in Figure 37 as                                   #in pCIB5515." contained                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - ATG GAC AAC AAC CCC AAC ATC AAC GAG TGC AT - #C CCC TAC AAC TGC CTG           48                                                                          Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu           #   420                                                                       - AGC AAC CCC GAG GTG GAG GTG CTG GGC GGC GA - #G CGC ATC GAG ACC GGC           96                                                                          Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly           425                 4 - #30                 4 - #35                 4 -       #40                                                                           - TAC ACC CCC ATC GAC ATC AGC CTG AGC CTG AC - #C CAG TTC CTG CTG AGC          144                                                                          Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser           #               455                                                           - GAG TTC GTG CCC GGC GCC GGC TTC GTG CTG GG - #C CTG GTG GAC ATC ATC          192                                                                          Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           #           470                                                               - TGG GGC ATC TTC GGC CCC AGC CAG TGG GAC GC - #C TTC CTG GTG CAG ATC          240                                                                          Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile           #       485                                                                   - GAG CAG CTG ATC AAC CAG CGC ATC GAG GAG TT - #C GCC CGC AAC CAG GCC          288                                                                          Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala           #   500                                                                       - ATC AGC CGC CTG GAG GGC CTG AGC AAC CTG TA - #C CAA ATC TAC GCC GAG          336                                                                          Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu           505                 5 - #10                 5 - #15                 5 -       #20                                                                           - AGC TTC CGC GAG TGG GAG GCC GAC CCC ACC AA - #C CCC GCC CTG CGC GAG          384                                                                          Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu           #               535                                                           - GAG ATG CGC ATC CAG TTC AAC GAC ATG AAC AG - #C GCC CTG ACC ACC GCC          432                                                                          Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala           #           550                                                               - ATC CCC CTG TTC GCC GTG CAG AAC TAC CAG GT - #G CCC CTG CTG AGC GTG          480                                                                          Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           #       565                                                                   - TAC GTG CAG GCC GCC AAC CTG CAC CTG AGC GT - #G CTG CGC GAC GTC AGC          528                                                                          Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser           #   580                                                                       - GTG TTC GGC CAG CGC TGG GGC TTC GAC GCC GC - #C ACC ATC AAC AGC CGC          576                                                                          Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg           585                 5 - #90                 5 - #95                 6 -       #00                                                                           - TAC AAC GAC CTG ACC CGC CTG ATC GGC AAC TA - #C ACC GAC CAC GCC GTG          624                                                                          Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val           #               615                                                           - CGC TGG TAC AAC ACC GGC CTG GAG CGC GTG TG - #G GGT CCC GAC AGC CGC          672                                                                          Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg           #           630                                                               - GAC TGG ATC AGG TAC AAC CAG TTC CGC CGC GA - #G CTG ACC CTG ACC GTG          720                                                                          Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           #       645                                                                   - CTG GAC ATC GTG AGC CTG TTC CCC AAC TAC GA - #C AGC CGC ACC TAC CCC          768                                                                          Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro           #   660                                                                       - ATC CGC ACC GTG AGC CAG CTG ACC CGC GAG AT - #T TAC ACC AAC CCC GTG          816                                                                          Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val           665                 6 - #70                 6 - #75                 6 -       #80                                                                           - CTG GAG AAC TTC GAC GGC AGC TTC CGC GGC AG - #C GCC CAG GGC ATC GAG          864                                                                          Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu           #               695                                                           - GGC AGC ATC CGC AGC CCC CAC CTG ATG GAC AT - #C CTG AAC AGC ATC ACC          912                                                                          Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr           #           710                                                               - ATC TAC ACC GAC GCC CAC CGC GGC GAG TAC TA - #C TGG AGC GGC CAC CAG          960                                                                          Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln           #       725                                                                   - ATC ATG GCC AGC CCC GTC GGC TTC AGC GGC CC - #C GAG TTC ACC TTC CCC         1008                                                                          Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro           #   740                                                                       - CTG TAC GGC ACC ATG GGC AAC GCT GCA CCT CA - #G CAG CGC ATC GTG GCA         1056                                                                          Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala           745                 7 - #50                 7 - #55                 7 -       #60                                                                           - CAG CTG GGC CAG GGA GTG TAC CGC ACC CTG AG - #C AGC ACC CTG TAC CGT         1104                                                                          Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg           #               775                                                           - CGA CCT TTC AAC ATC GGC ATC AAC AAC CAG CA - #G CTG AGC GTG CTG GAC         1152                                                                          Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp           #           790                                                               - GGC ACC GAG TTC GCC TAC GGC ACC AGC AGC AA - #C CTG CCC AGC GCC GTG         1200                                                                          Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           #       805                                                                   - TAC CGC AAG AGC GGC ACC GTG GAC AGC CTG GA - #C GAG ATC CCC CCT CAG         1248                                                                          Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln           #   820                                                                       - AAC AAC AAC GTG CCA CCT CGA CAG GGC TTC AG - #C CAC CGT CTG AGC CAC         1296                                                                          Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His           825                 8 - #30                 8 - #35                 8 -       #40                                                                           - GTG AGC ATG TTC CGC AGT GGC TTC AGC AAC AG - #C AGC GTG AGC ATC ATC         1344                                                                          Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile           #               855                                                           - CGT GCA CCT ATG TTC AGC TGG ATT CAC CGC AG - #T GCC GAG TTC AAC AAC         1392                                                                          Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn           #           870                                                               - ATC ATC CCC AGC AGC CAG ATC ACC CAG ATC CC - #C CTG ACC AAG AGC ACC         1440                                                                          Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           #       885                                                                   - AAC CTG GGC AGC GGC ACC AGC GTG GTG AAG GG - #C CCC GGC TTC ACC GGC         1488                                                                          Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly           #   900                                                                       - GGC GAC ATC CTG CGC CGC ACC AGC CCC GGC CA - #G ATC AGC ACC CTG CGC         1536                                                                          Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg           905                 9 - #10                 9 - #15                 9 -       #20                                                                           - GTG AAC ATC ACC GCC CCC CTG AGC CAG CGC TA - #C CGC GTC CGC ATC CGC         1584                                                                          Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg           #               935                                                           - TAC GCC AGC ACC ACC AAC CTG CAG TTC CAC AC - #C AGC ATC GAC GGC CGC         1632                                                                          Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg           #           950                                                               - CCC ATC AAC CAG GGC AAC TTC AGC GCC ACC AT - #G AGC AGC GGC AGC AAC         1680                                                                          Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           #       965                                                                   - CTG CAG AGC GGC AGC TTC CGC ACC GTG GGC TT - #C ACC ACC CCC TTC AAC         1728                                                                          Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn           #   980                                                                       - TTC AGC AAC GGC AGC AGC GTG TTC ACC CTG AG - #C GCC CAC GTG TTC AAC         1776                                                                          Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn           985                 9 - #90                 9 - #95                 1 -       #000                                                                          - AGC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTG CCC GCC GAG         1824                                                                          Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu           #              10150                                                          - GTG ACC TTC GAG GCC GAG TAC GAC CTG GAG AG - #G GCT CAG AAG GCC GTG         1872                                                                          Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val           #          10305                                                              - AAC GAG CTG TTC ACC AGC AGC AAC CAG ATC GG - #C CTG AAG ACC GAC GTG         1920                                                                          Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val           #      10450                                                                  - ACC GAC TAC CAC ATC GAT CAA GTA TCC AAT TT - #A GTT GAG TGT TTA TCT         1968                                                                          Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser           #  10605                                                                      - GAT GAA TTT TGT CTG GAT GAA AAA AAA GAA TT - #G TCC GAG AAA GTC AAA         2016                                                                          Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys           #               10801070 - #                1075                              - CAT GCG AAG CGA CTT AGT GAT GAG CGG AAT TT - #A CTT CAA GAT CCA AAC         2064                                                                          His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn           #              10950                                                          - TTT AGA GGG ATC AAT AGA CAA CTA GAC CGT GG - #C TGG AGA GGA AGT ACG         2112                                                                          Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr           #          11105                                                              - GAT ATT ACC ATC CAA GGA GGC GAT GAC GTA TT - #C AAA GAG AAT TAC GTT         2160                                                                          Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val           #      11250                                                                  - ACG CTA TTG GGT ACC TTT GAT GAG TGC TAT CC - #A ACG TAT TTA TAT CAA         2208                                                                          Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln           #  11405                                                                      - AAA ATA GAT GAG TCG AAA TTA AAA GCC TAT AC - #C CGT TAC CAA TTA AGA         2256                                                                          Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg           #               11601150 - #                1155                              - GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA AT - #C TAT TTA ATT CGC TAC         2304                                                                          Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr           #              11750                                                          - AAT GCC AAA CAC GAA ACA GTA AAT GTG CCA GG - #T ACG GGT TCC TTA TGG         2352                                                                          Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp           #          11905                                                              - CCG CTT TCA GCC CCA AGT CCA ATC GGA AAA TG - #T GGG GAG CCG AAT CGA         2400                                                                          Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg           #      12050                                                                  - TGC GCT CCG CAC CTG GAG TGG AAC CCG GAC CT - #A GAC TGC AGC TGC AGG         2448                                                                          Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg           #  12205                                                                      - GAC GGG GAG AAG TGC GCC CAT CAT TCC CAT CA - #T TTC TCC TTG GAC ATT         2496                                                                          Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile           #               12401230 - #                1235                              - GAT GTT GGA TGT ACA GAC TTA AAT GAG GAC TT - #A GGT GTA TGG GTG ATA         2544                                                                          Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile           #              12550                                                          - TTC AAG ATT AAG ACG CAA GAT GGC CAT GCA AG - #A CTA GGA AAT CTA GAA         2592                                                                          Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu           #          12705                                                              - TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GC - #A CTA GCT CGT GTG AAA         2640                                                                          Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys           #      12850                                                                  - AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GA - #A AAA TTG GAA TGG GAA         2688                                                                          Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu           #  13005                                                                      - ACA AAT ATT GTT TAT AAA GAG GCA AAA GAA TC - #T GTA GAT GCT TTA TTT         2736                                                                          Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe           #               13201310 - #                1315                              - GTA AAC TCT CAA TAT GAT AGA TTA CAA GCG GA - #T ACC AAC ATC GCG ATG         2784                                                                          Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met           #              13350                                                          - ATT CAT GCG GCA GAT AAA CGC GTT CAT AGC AT - #T CGA GAA GCT TAT CTG         2832                                                                          Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu           #          13505                                                              - CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GC - #G GCT ATT TTT GAA GAA         2880                                                                          Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu           #      13650                                                                  - TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CT - #A TAT GAT GCG AGA AAT         2928                                                                          Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn           #  13805                                                                      - GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TT - #A TCC TGC TGG AAC GTG         2976                                                                          Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val           #               14001390 - #                1395                              - AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AA - #C CAC CGT TCG GTC CTT         3024                                                                          Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu           #              14150                                                          - GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CA - #A GAA GTT CGT GTC TGT         3072                                                                          Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys           #          14305                                                              - CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GC - #G TAC AAG GAG GGA TAT         3120                                                                          Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr           #      14450                                                                  - GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GA - #G AAC AAT ACA GAC GAA         3168                                                                          Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu           #  14605                                                                      - CTG AAG TTT AGC AAC TGT GTA GAA GAG GAA GT - #A TAT CCA AAC AAC ACG         3216                                                                          Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr           #               14801470 - #                1475                              - GTA ACG TGT AAT GAT TAT ACT GCG ACT CAA GA - #A GAA TAT GAG GGT ACG         3264                                                                          Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr           #              14950                                                          - TAC ACT TCT CGT AAT CGA GGA TAT GAC GGA GC - #C TAT GAA AGC AAT TCT         3312                                                                          Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser           #          15105                                                              - TCT GTA CCA GCT GAT TAT GCA TCA GCC TAT GA - #A GAA AAA GCA TAT ACA         3360                                                                          Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr           #      15250                                                                  - GAT GGA CGA AGA GAC AAT CCT TGT GAA TCT AA - #C AGA GGA TAT GGG GAT         3408                                                                          Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp           #  15405                                                                      - TAC ACA CCA CTA CCA GCT GGC TAT GTG ACA AA - #A GAA TTA GAG TAC TTC         3456                                                                          Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe           #               15601550 - #                1555                              - CCA GAA ACC GAT AAG GTA TGG ATT GAG ATC GG - #A GAA ACG GAA GGA ACA         3504                                                                          Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr           #              15750                                                          - TTC ATC GTG GAC AGC GTG GAA TTA CTT CTT AT - #G GAG GAA TAA                 #3546                                                                         Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                       #           1585                                                              - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1181 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu         #                 15                                                          - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly         #             30                                                              - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser         #         45                                                                  - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile         #     60                                                                      - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile         # 80                                                                          - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala         #                 95                                                          - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu         #           110                                                               - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu         #       125                                                                   - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala         #   140                                                                       - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser         #               175                                                           - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg         #           190                                                               - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp His Ala Val         #       205                                                                   - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg         #   220                                                                       - Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr As - #p Ser Arg Thr Tyr Pro         #               255                                                           - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val         #           270                                                               - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu         #       285                                                                   - Gly Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr         #   300                                                                       - Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty - #r Trp Ser Gly His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro         #               335                                                           - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala         #           350                                                               - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg         #       365                                                                   - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp         #   380                                                                       - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln         #               415                                                           - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His         #           430                                                               - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile         #       445                                                                   - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn         #   460                                                                       - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly         #               495                                                           - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg         #           510                                                               - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg         #       525                                                                   - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg         #   540                                                                       - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn         #               575                                                           - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn         #           590                                                               - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu         #       605                                                                   - Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Ar - #g Ala Gln Lys Ala Val         #   620                                                                       - Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gl - #y Leu Lys Thr Asp Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Thr Asp Tyr His Ile Asp Gln Val Ser Asn Le - #u Val Glu Cys Leu Ser         #               655                                                           - Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Le - #u Ser Glu Lys Val Lys         #           670                                                               - His Ala Lys Arg Leu Ser Asp Glu Arg Asn Le - #u Leu Gln Asp Pro Asn         #       685                                                                   - Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gl - #y Trp Arg Gly Ser Thr         #   700                                                                       - Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Ph - #e Lys Glu Asn Tyr Val         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pr - #o Thr Tyr Leu Tyr Gln         #               735                                                           - Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Th - #r Arg Tyr Gln Leu Arg         #           750                                                               - Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Il - #e Tyr Leu Ile Arg Tyr         #       765                                                                   - Asn Ala Lys His Glu Thr Val Asn Val Pro Gl - #y Thr Gly Ser Leu Trp         #   780                                                                       - Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cy - #s Gly Glu Pro Asn Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Cys Ala Pro His Leu Glu Trp Asn Pro Asp Le - #u Asp Cys Ser Cys Arg         #               815                                                           - Asp Gly Glu Lys Cys Ala His His Ser His Hi - #s Phe Ser Leu Asp Ile         #           830                                                               - Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Le - #u Gly Val Trp Val Ile         #       845                                                                   - Phe Lys Ile Lys Thr Gln Asp Gly His Ala Ar - #g Leu Gly Asn Leu Glu         #   860                                                                       - Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Al - #a Leu Ala Arg Val Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Gl - #u Lys Leu Glu Trp Glu         #               895                                                           - Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Se - #r Val Asp Ala Leu Phe         #           910                                                               - Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala As - #p Thr Asn Ile Ala Met         #       925                                                                   - Ile His Ala Ala Asp Lys Arg Val His Ser Il - #e Arg Glu Ala Tyr Leu         #   940                                                                       - Pro Glu Leu Ser Val Ile Pro Gly Val Asn Al - #a Ala Ile Phe Glu Glu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Le - #u Tyr Asp Ala Arg Asn         #               975                                                           - Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Le - #u Ser Cys Trp Asn Val         #           990                                                               - Lys Gly His Val Asp Val Glu Glu Gln Asn As - #n His Arg Ser Val Leu         #      10050                                                                  - Val Val Pro Glu Trp Glu Ala Glu Val Ser Gl - #n Glu Val Arg Val Cys         #  10205                                                                      - Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Al - #a Tyr Lys Glu Gly Tyr         #               10401030 - #                1035                              - Gly Glu Gly Cys Val Thr Ile His Glu Ile Gl - #u Asn Asn Thr Asp Glu         #              10550                                                          - Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Va - #l Tyr Pro Asn Asn Thr         #          10705                                                              - Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Gl - #u Glu Tyr Glu Gly Thr         #      10850                                                                  - Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Al - #a Tyr Glu Ser Asn Ser         #  11005                                                                      - Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Gl - #u Glu Lys Ala Tyr Thr         #               11201110 - #                1115                              - Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser As - #n Arg Gly Tyr Gly Asp         #              11350                                                          - Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe         #          11505                                                              - Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Thr         #      11650                                                                  - Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                     #  11805                                                                      - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 88 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE74A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - GCAGATCTGG ATCCATGCAC GCCGTGAAGG GCCCTTCTAG AAGGCCTATC GA - #TAAAGAGC         60                                                                          #             88   GCAC GCAGGTTC                                              - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE72A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #    40            TCAG AAGAACTCGT CAAGAAGGCG                                 - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P1(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #                 22AAT GG                                                    - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P1(b)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #              26  CCAA CAATGG                                                - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P2(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #                23CAGC ACG                                                   - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P2(b)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #                23CGCG CAG                                                   - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer A1"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer A2"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P3(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #                24GTGT TCGG                                                  - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P3(b)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #                23CAGC GTG                                                   - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P4(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #               25 CCGT ACAGG                                                 - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P4(b)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #                23GGTG CCG                                                   - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer B1"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer B2"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P5(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #          32      CCAT GGGCAACGCC GC                                         - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P5(b)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                #                23GGGC AAC                                                   - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P6(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                #              26  ACCA CGCTGG                                                - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P6(b)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #                23CTTC ACC                                                   - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer C1"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 13 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                   first half"2 DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                   second half" DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                   forward"r PEPCivs#9 TION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                # 19               CTC                                                        - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer PEPCivs#9 reverse"esc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                # 19               AGT                                                        - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P7(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #               25 CTTC ACCGG                                                 - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer P8(a)"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #       36         TACA CCTGATCGAT GTGGTA                                     - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer for fourth quarter sc                                                             second ha - #lf"                                               -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                # 20               TGAT                                                       - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 11 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer for third quarter esc                                                             first hal - #f"                                                -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer MK23A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                # 20               CCCT                                                       - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer MK25A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                # 20               TGCT                                                       - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer MK26A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                # 20               TGAA                                                       - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "sequence in pCIB3073 priorsc                                                             to deleti - #on"                                               -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                #         33       GCAA GATCTGAGAT ATG                                        - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE134A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                # 44               ATCG ATCAAGTATC CAATTTAGTT GAGT                            - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE135A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                # 44               TACT TGATCGATGT GGTAGTCGGT CACG                            - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE136A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                #      37          AGGT ACCCAATAGC GTAACGT                                    - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE137A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #21                CCTA T                                                     - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE138A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                #     38           ATTC CTCCATAAGA AGTAATTC                                   - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer MK05A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #                23CGTA ACG                                                   - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer MK35A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                # 20               ACCG                                                       - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "forward primer forION: /desc                                                             pCIB4434"                                                      -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                #  42              CCAA GGAGGCGATG ACGTATTCAA AG                              - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "reverse primer forION: /desc                                                             pCIB4434"                                                      -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                #             51GGCTCCC CGCACTTGCC GATTGGACTT GGGGCTGAAA G                    - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #1"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                #           30     GGGT ACCTTTGATG                                            - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 98 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #2"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                - TCCCCGTCCC TGCAGCTGCA GTCTAGGTCC GGGTTCCACT CCAGGTGCGG AG - #CGCATCGA         60                                                                          #     98           TGCC GATTGGACTT GGGGCTGA                                   - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 98 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #3"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                - CAAGTGCGGG GAGCCGAATC GATGCGCTCC GCACCTGGAG TGGAACCCGG AC - #CTAGACTG         60                                                                          #     98           GAAA AATGTGCCCA TCATTCCC                                   - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #4"DESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                #           30     AATT CTAGATTTCC                                            - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer used to mapION: /desc                                                             transcriptio - #nal start site for TrpA gene"                  -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                #                24CGTC GAGG                                                  - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -      (v) FRAGMENT TYPE: N-terminal                                          -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..26                                                 #/note= "N-terminal peptide from                                                             pollen sp - #ecific protein"                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                - Thr Thr Pro Leu Thr Phe Gln Val Gly Lys Gl - #y Ser Lys Pro Gly His         #                15                                                           - Leu Ile Leu Thr Pro Asn Val Ala Thr Ile                                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..20                                                 #/note= "internal peptide of pollen                                           #protein"      specific                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                - Lys Pro Gly His Leu Ile Leu Thr Pro Asn Va - #l Ala Thr Ile Ser Asp         #                15                                                           - Val Val Ile Lys                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..16                                                 #/note= "internal peptide fromN:                                                             pollen sp - #ecific protein"                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                - Ser Gly Gly Thr Arg Ile Ala Asp Asp Val Il - #e Pro Ala Asp Phe Lys         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..12                                                 #/note= "internal peptide fromN:                                                             pollen sp - #ecific protein"                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                - Glu His Gly Gly Asp Asp Phe Ser Phe Thr Le - #u Lys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -      (v) FRAGMENT TYPE: internal                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..12                                                 #/note= "internal peptide fromN:                                                             pollen sp - #ecific protein"                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                - Glu Gly Pro Thr Gly Thr Trp Thr Leu Asp Th - #r Lys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "oligonucleotide #51"N: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                # 20               GYTC                                                       - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "oligonucleotide #58"N: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "oligonucleotide PE51": /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                #         33       CGGG GAACGAGTGC GGC                                        - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #42"ESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                #    40            GCAT GCGATCTGCA CCTCCCGCCG                                 - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #43"ESCRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                #  18              GG                                                         - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #SK50"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                #21                AACC T                                                     - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer #SK49"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                #             27   CGAG AGATGGA                                               - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE99A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                #                 22ACA TG                                                    - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE97A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                #   41             GCGG ATCCCGCGGC GGGAAGCTAA G                               - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE100A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE98A28"IPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                #    39            GCGG ATCCTGTCCG ACACCGGAC                                  - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE104A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                # 20               ACAC                                                       - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE103A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                #       35         TGTC CGACACCGGA CGGCT                                      - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE127"CRIPTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                #              26  CGGG GAACGA                                                - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE150A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                #                23CATT ATC                                                   - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer KE151A28"PTION: /desc                                              -    (iii) HYPOTHETICAL: NO                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                #      37          CGTC GCTTCTGTGC AACAACC                                    __________________________________________________________________________

What is claimed is:
 1. A nucleic acid molecule comprising a 1.8 kbpromoter fragment isolated from the 5' flanking region upstream of thestart site of the coding region of a plant tryptophan synthase alphasubunit (trpA) gene, wherein said promoter fragment is capable ofdirecting pith-preferred expression of an associated coding sequence ina plant.
 2. A nucleic acid molecule according to claim 1, wherein saidplant trpA gene is a maize trpA gene.
 3. A nucleic acid moleculeaccording to claim 2, wherein said promoter fragment is isolated fromthe region upstream of nucleotide number 1839 of SEQ ID NO:18.
 4. Anucleic acid molecule according to claim 2, wherein said promoterfragment comprises nucleotides 39-1838 of SEQ ID NO:18.
 5. A chimericDNA molecule comprising the nucleic acid molecule according to claim 1operatively linked to a coding sequence of interest.
 6. A chimeric DNAmolecule comprising the nucleic acid molecule according to claim 2operatively linked to a coding sequence of interest.
 7. A chimeric DNAmolecule according to claim 6, wherein the coding sequence of interestis the coding sequence of a maize trpA gene.
 8. A chimeric DNA moleculeaccording to claim 6, wherein the coding sequence of interest is thecoding sequence of an insecticidal protein gene.
 9. A chimeric DNAmolecule according to claim 8, wherein the coding sequence of interestis the coding sequence of a Bacillus thuringiensis protein gene.
 10. Aplant transformation vector comprising the chimeric DNA moleculeaccording to claim
 5. 11. A plant transformation vector comprising thechimeric DNA molecule according to claim
 6. 12. A plant transformationvector according to claim 11, which is plasmid pCIB4433 (NRRL B-18999).13. A transgenic plant or plant part comprising the chimeric DNAmolecule according to claim
 5. 14. A transgenic plant or plant partcomprising the chimeric DNA molecule according to claim
 6. 15. Atransgenic plant or plant part according to claim 14, which is maize.